Comparative profiling of rhizosphere bacterial communities in male and female date palms reveals sex-associated taxonomic shifts using 16S rRNA amplicon sequencing

The rhizosphere microbiome plays a vital role in plant nutrition, health, and resilience, supporting sustainable agriculture and contributing to SDG 2 (Zero Hunger) and SDG 12 (Responsible Consumption and Production). However, comparative studies of rhizosphere bacterial communities associated with male and female date palm (Phoenix dactylifera L.) plants remain relatively limited. Therefore, this study aimed to compare the rhizosphere bacterial communities associated with male and female date palm plants. Rhizosphere bacterial communities associated with male and female date palm plants were investigated using 16S rRNA gene amplicon sequencing to evaluate differences in bacterial community composition and diversity associated with plant sex. Across both sexes, the rhizosphere microbiome was dominated by Firmicutes, Proteobacteria, Actinobacteriota, Acidobacteriota, and Chloroflexi, with Bacillus -related taxa prominent at lower taxonomic levels. Shannon diversity and observed features did not differ significantly between female and male samples (p = 0.1266 and p = 0.13, respectively). In contrast, beta diversity analysis revealed no significant separation between male and female communities, indicating similar overall community structure. Although exploratory differences in relative abundance were observed for certain taxa (Bdellovibrionota, Entotheonellaeota, Planctomycetota, Micromonosporales, Thermomicrobiales, Pedosphaerales, and Chitinophagales). Male and female date palm plants harbor broadly similar rhizosphere bacterial communities with analyzing differences in selected taxa. These preliminary observations warrant further investigation with larger sample sizes, functional approaches, and explicit consideration of reproductive stage effects, with potential implications for developing microbiome-informed strategies to enhance soil health and sustainable date palm productivity.

The rhizosphere represents a highly dynamic ecological interface where plant roots interact with diverse microbial communities. This narrow zone of soil surrounding plant roots harbours complex assemblage of bacteria, fungi, and other microorganisms that play crucial roles in plant growth, nutrient cycling, and stress tolerance (Feng et al., 2026). Rhizosphere microorganisms can enhance plant productivity through multiple mechanisms, including nitrogen fixation, phosphorus solubilization, phytohormone production, and protection against soil-borne pathogens (Singh et al., 2024; Thepbandit and Athinuwat, 2024). Consequently, understanding the composition and dynamics of rhizosphere microbial communities has become an important focus in plant-microbe interaction studies (Abubakari and Abubakari, 2026). Plants actively shape the structure and diversity of rhizosphere microbial communities through the release of root exudates, which include sugars, amino acids, organic acids, and secondary metabolites. These compounds serve as substrates and signaling molecules that selectively recruit specific microbial taxa from the surrounding soil environment (Martins et al., 2026). Several studies have demonstrated that plant species, genotype, developmental stage, and environmental conditions can significantly influence rhizosphere microbiome composition (Negre Rodríguez et al., 2025; Liu et al., 2025). However, the role of plant sex in shaping rhizosphere microbial communities remains relatively unexplored, particularly in dioecious plant species (Guo et al., 2022).

Dioecious plants, which possess separate male and female individuals, often exhibit physiological, biochemical, and ecological differences between sexes. Female plants, particularly during reproductive and fruiting stages, may experience increased nutrient demand and altered root exudation, potentially leading to higher microbial diversity, different community composition, and selective enrichment of specific functional groups compared to males. These differences may influence root exudation patterns, nutrient uptake, and plant-microbe interactions, ultimately affecting the structure of rhizosphere microbial communities (Xia et al., 2022). Previous studies in certain dioecious species have suggested that plant sex can influence soil microbial diversity and community composition, indicating that male and female plants may recruit distinct microbial assemblages (Yan et al., 2025; Liao et al., 2026). Nevertheless, such sex-specific microbial associations have rarely been investigated in economically important perennial crops.

Date palm (Phoenix dactylifera L.) is a dioecious perennial tree of significant agricultural and economic importance, particularly in arid and semi-arid regions of the Middle East, North Africa, and South Asia (Amjad et al., 2026). In date palm cultivation, male and female plants differ not only in reproductive function but also in physiological and metabolic characteristics. These differences may potentially influence rhizosphere microbial communities. Since reproductive stage can independently influence rhizosphere microbiota, true sex-dependent effects require sampling male and female plants at equivalent developmental stages or explicitly including reproductive stage as a variable in analyses. The composition and diversity of microbiota associated with male and female date palms remain relatively underexplored. Advances in high-throughput sequencing technologies have enabled detailed characterization of microbial communities in environmental samples. Among these approaches, 16S rRNA gene amplicon sequencing is widely used to investigate bacterial diversity and community structure in the rhizosphere, providing valuable insights into plant-associated microbiomes (Ben Zineb et al., 2024; Rahman et al., 2025).

Understanding rhizosphere microbial variation associated with plant sex is important for improving crop productivity and sustainability, particularly in dioecious perennial crops such as date palm. Insights into sex-associated microbial variation can contribute to the development of targeted microbiome-based strategies for enhancing nutrient use efficiency, soil health, and plant resilience under arid and semi-arid conditions. In this context, the present study aimed to compare the rhizosphere bacterial communities associated with male and female date palm (Phoenix dactylifera L.) plants using high throughput 16S rRNA gene sequencing. To evaluate sex-associated patterns while acknowledging potential developmental-stage confounding, microbial diversity, community structure, and taxonomic abundance were analysed to examine potential sex-associated variation in the rhizosphere bacterial community. Collectively, this study provides fundamental insight into sex-dependent plant-microbe interactions in date palm and establishes a foundation for future applications in sustainable crop management.

Sample collection

Rhizosphere soil samples were collected from healthy male and female date palm (Phoenix dactylifera L.) plants of the "Barhee" cultivar, approximately 10 years of age and comparable tree size based on visual field assessment located at three different sites in Gujarat, India. Sampling was conducted on the same day, during the fruiting stage of female plants while male plants were in post-flowering stage. The sampling locations included Juna Sadulka (22.877733° N, 70.809763° E), Khevaliya (22.828667° N, 70.685546° E), and Morbi (22.695206° N, 70.707872° E). At each location, rhizosphere samples were collected from both male and female date palm plants. For each plant sex, rhizosphere soil from five individual plants was pooled to form a composite sample to reduce variability among individual plants within the same site. All selected farms were organically fertilized with cow-dung compost and maintained under adequate, regular irrigation using available ground water. The soils from the three sampling sites were generally alkaline and predominantly sandy, with a mean pH of 7.87 ± 0.05, electrical conductivity (EC) of 10.40 ± 0.10 dS m −1, organic carbon of 0.35% ± 0.02%, total nitrogen of 2.64% ± 0.05%, available phosphorus of 49.52 ± 0.53 kg ha −1, exchangeable potassium of 1.58 ± 0.06 ppm, and water-holding capacity of 32.00% ± 2.00% in the soil layer.

In total, six composite samples were obtained, including three female samples (F1–F3) and three male samples (M4–M6), each representing pooled rhizosphere soil from five plants at a given location. The five individual plants contributing to each composite sample were not treated as independent statistical replicates in subsequent analyses. Therefore, the effective biological replication for all statistical analyses was n = 3 per sex. This limited replication constrains statistical power and the ability to infer generalized plant-sex effects across broader date palm populations. For sample collection, the surface soil was carefully removed to expose the root zone, and rhizosphere soil was collected from approximately 15 cm depth. Soil tightly adhering to the root surface was gently collected using sterile tools. The rhizosphere soil was placed in sterile containers and transported to the laboratory for further molecular analysis.

DNA extraction and 16S rRNA gene sequencing

Total genomic DNA was extracted from rhizosphere soil samples using a soil DNA extraction kit (DNeasy Power Soil Pro, Qiagen) suitable for environmental samples. The V3–V4 hypervariable region of the bacterial 16S rRNA gene was amplified using the forward primer 16S rRNA F (5′-GCCTACGGGNGGCWGCAG-3′) and reverse primer 16S rRNA R (5′-GACTACHVGGGTATCTAATCC-3′), incorporating Illumina overhang adapter sequences. PCR amplification was performed in a final volume of 25 µL containing 12.5 µL of 2 × KAPA HiFi HotStart ReadyMix (Roche, Basel, Switzerland), 1 µL of forward primer (10 µM), 1 µL of reverse primer (10 µM), and 10.5 µL of template DNA (30 ng total). Thermal cycling consisted of an initial denaturation at 95 °C for 5 min, followed by 28 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 45 s, followed by a final extension at 72 °C for 7 min and a hold at 4 °C. The resulting amplicons were purified using AMPure XP magnetic beads to remove residual primers, primer-dimers, and other small DNA fragments. The expected amplicon size was approximately 550 bp, including the Illumina overhang adapter sequences.

In a subsequent indexing PCR, dual-index barcodes and Illumina sequencing adapters were added using the Nextera XT Indexing strategy, enabling multiplex sequencing of samples. Each indexing PCR was performed in a final reaction volume of 25 µL containing 12.5 µL of 2 × KAPA HiFi HotStart ReadyMix (Roche, Basel, Switzerland), 5 µL each of Nextera XT Index Primer 1 (i7) and Index Primer 2 (i5), 7.5 µL of purified amplicon DNA (100ng total). Amplification was carried out under the following thermal cycling conditions: an initial denaturation at 95 °C for 5 min, followed by 8 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 45 s, with a final extension at 72 °C for 7 min and a hold at 4 °C. The indexed libraries were subsequently purified using AMPure XP magnetic beads to remove residual primers, primer dimers, and short amplification products. Purified libraries were assessed for quality and quantified using fluorometric methods, and the expected library size was approximately 630 bp, including index and adapter sequences. The libraries were quantified, normalized, and pooled at equimolar concentrations prior to sequencing. Sequencing was performed on the Illumina MiSeq platform using the MiSeq Reagent Kit v3 with paired-end 2 × 300 bp chemistry. The resulting paired-end reads were merged to generate high-quality consensus sequences spanning the V3–V4 region of the 16S rRNA gene for downstream microbial community analysis. The raw sequencing data have been deposited in the European Nucleotide Archive (ENA) under accession number PRJEB111153.

Bioinformatics and sequence processing

Raw sequencing reads obtained from the Illumina MiSeq platform were processed using the QIIME2 (Quantitative Insights Into Microbial Ecology 2) bioinformatics pipeline (qiime2-amplicon-2024.2) (Estaki et al., 2020). Adapter sequences, ambiguous reads, and low-quality sequences were removed using Trimmomatic (v0.38) (Bolger et al., 2014) with the following parameters: adapter removal (ILLUMINACLIP:2:30:10), leading and trailing base trimming (LEADING:20, TRAILING:20), sliding-window quality filtering (SLIDINGWINDOW:20:25), and a minimum read length of 100 bp (MINLEN:100). Paired-end reads were merged using FLASH (v1.2.11) (Magoč and Salzberg, 2011), with a minimum overlap length of 40 bp to ensure high-confidence sequence merging. Quality-filtered reads were then subjected to DADA2 denoising using the QIIME 2 DADA2 denoise-single plugin with the following parameters: no additional truncation (truncLen = 0), default ASV inference algorithm, maximum expected error (maxEE) of 2.0, and truncation quality threshold (truncQ) of 2. Chimeric sequences were removed using the consensus method. Taxonomic classification of amplicon sequence variants (ASVs) was performed using the q2-feature-classifier with a Naive Bayes classifier and pre-trained SILVA database release 138.1 reference database (trained at 99% sequence identity threshold for 16S rRNA gene sequences) (Quast et al., 2013). High-quality ASVs and their taxonomic classifications were used for downstream alpha-diversity, beta-diversity, and comparative abundance analyses. Both alpha- and beta-diversity analyses were performed using a rarefied feature table. Specifically, the filtered ASV feature table was rarefied to a sampling depth of 5,000 sequences per sample using the QIIME 2 core-metrics-phylogenetic workflow. Thus, samples were subsampled to an equal sequencing depth of 5,000 sequences prior to calculation of the alpha-diversity and beta-diversity metrics.

Taxonomic composition analysis

Taxonomic composition of the rhizosphere microbial communities was analysed at multiple taxonomic levels; including phylum, class, order, family, and genus. Relative abundance of bacterial taxa was calculated for each sample and visualized using stacked bar plots and heatmaps to compare microbial community profiles between male and female rhizosphere samples. In addition, a comparative abundance analysis was performed using the mean relative abundance of taxa for female and male groups. The Relative-abundance difference (%) between the groups were calculated as per the given formula to assess sex-associated variation in microbial taxa. This metric expresses the proportional difference relative to the female reference.

Microbial diversity and statistical analysis

Microbial diversity within individual samples (alpha diversity) was evaluated using the Shannon diversity index to assess microbial richness and evenness. Differences in alpha diversity between male and female rhizosphere samples were tested using the Kruskal–Wallis test. To examine differences in microbial community composition between samples, beta diversity analysis was performed using weighted and unweighted UniFrac distance metrics (Lozupone and Knight, 2005). Weighted UniFrac incorporates both the presence/absence and relative abundance of taxa, while unweighted UniFrac considers only presence/absence patterns. Principal coordinate analysis (PCoA) was used to visualize patterns of similarity and dissimilarity among microbial communities. The statistical significance of differences in microbial community structure between groups was assessed using permutational multivariate analysis of variance (PERMANOVA) (Anderson, 2001), with plant sex (female vs. male) as the grouping factor and 999 permutations. Sampling location was not included in the PERMANOVA design. Tests of homogeneity of multivariate dispersion were not performed. In addition, linear discriminant analysis effect size (LEfSe) was used to identify bacterial taxa that were significantly enriched in either male or female rhizosphere samples (Segata et al., 2011). Taxa were considered discriminant when they met an LDA effect-size threshold of ≥2.0 and a Kruskal–Wallis statistical significance threshold of p < 0.05.

Sequencing output and microbial diversity coverage

High-throughput sequencing of rhizosphere soil samples generated a substantial number of reads across all samples (Table 1). After quality filtering, high-quality reads ranged from 56,654 to 92,555 per sample, with corresponding high-quality sequence data ranging from 29.01 MB to 47.63 MB, indicating adequate sequencing depth for downstream microbial community analysis. These results are consistent with earlier rhizosphere microbiome studies, where comparable sequencing depths have been shown to provide adequate coverage for downstream analyses (Laili et al., 2025). Rarefaction analysis further confirmed that sequencing depth was adequate, as the rarefaction curves approached saturation for all samples, suggesting that most of the bacterial diversity present in the rhizosphere samples was captured consistent with previous studies where rarefaction curves indicated sufficient sequencing coverage of microbial communities (Long et al., 2025).

Alpha diversity of rhizosphere microbial communities

Alpha diversity analysis was performed to evaluate microbial richness and diversity within rhizosphere samples associated with male and female date palm plants. The number of observed features ranged from 1,078 to 1,990 across samples. Among female samples, F1 exhibited the highest number of observed features (1,990), followed by F3 (1,714) and F2 (1,647). Male samples showed comparatively lower richness, with observed features ranging from 1,078 in M6 to 1,701 in M5, while M4 contained 1,343 observed features. Similarly, the Shannon diversity index ranged from 8.44 to 9.50. The highest Shannon diversity was observed in F1 (9.50), followed by F2 (9.10) and F3 (9.00), whereas male samples showed Shannon index values of 8.68, 8.73, and 8.44 for M4, M5, and M6, respectively. Statistical analysis using the Kruskal–Wallis test indicated that differences in observed features did not differ between male and female samples (H = 2.33, p = 0.1266, q = 0.1266).

Taxonomic composition of rhizosphere microbial communities

The taxonomic composition of rhizosphere bacterial communities associated with male and female date palm plants was analysed across multiple taxonomic levels, including phylum, class, order, family, and genus. The individual sample-level relative abundance data for the different taxonomic levels are provided in Supplementary Table 1. At the phylum level (Supplementary Figures 1–3), rhizosphere microbial communities were dominated by Firmicutes, Proteobacteria, Actinobacteriota, Acidobacteriota, and Chloroflexi across all samples. In female rhizosphere samples, Proteobacteria represented the most relative abundant phylum in sample F1 (22.72%), followed by Firmicutes (17.85%) and Actinobacteriota (17.65%). Male rhizosphere samples also showed high relative abundance of dominant phyla, with Proteobacteria ranging from 15.15% to 23.25% and Firmicutes ranging from 17.99% to 37.70% across the samples. Other important phyla detected in the rhizosphere microbiome included Actinobacteriota (10.27%–17.73%), Acidobacteriota (7.32%–11.01%) and Chloroflexi (3.11%–8.65%).

At the class level (Supplementary Figures 4–6), Bacilli comprised the most relative abundant bacterial class across samples, ranging from 15.74% to 35.16%. Female samples showed Bacilli relative abundance values of 17.15% (F1), 24.44% (F2), and 27.90% (F3), whereas male samples exhibited 15.96% (M4), 35.16% (M5), and 15.75% (M6). Other class included Alphaproteobacteria ranged from 9.37% to 13.90%, while Gammaproteobacteria ranged from 5.78% to 9.48%. Actinobacteria ranged from 4.01% to 9.76%. Additional bacterial classes including Vicinamibacteria, Acidimicrobiia, Bacteroidia, Chloroflexia, Thermoleophilia, and Blastocatellia were also detected.

At the order level (Supplementary Figures 7–9), Bacillales represented the highest relative abundance order across samples, ranging from 9.48% to 23.25%. Female rhizosphere samples showed Bacillales relative abundance values of 11.00% (F1), 16.29% (F2), and 23.25% (F3), whereas male samples showed 9.73% (M4), 23.07% (M5), and 9.48% (M6). Additional bacterial orders included Vicinamibacteriales, Paenibacillales, Burkholderiales, Sphingomonadales, Tistrellales, and Rhizobiales. Further taxonomic classification at the family level (Supplementary Figures 10 and 11) revealed the presence of several bacterial families. Among these, Bacillaceae represented one of the dominant bacterial families, ranging from approximately 8.40% to 21.28% across the samples. Female rhizosphere samples showed Bacillaceae relative abundance values of 9.61% (F1), 14.77% (F2), and 20.97% (F3), whereas male samples showed 8.40% (M4), 21.28% (M5), and 8.80% (M6). In addition to Bacillaceae, several other bacterial families were detected, including Paenibacillaceae, Vicinamibacteraceae, Sphingomonadaceae, Geminicoccaceae, Rubrobacteriaceae, and Gemmatimonadaceae among others.

At the genus level (Supplementary Figures 12 and 13), Bacillus emerged as the most relative abundance genus in both male and female samples. Relative abundance ranged from 8.28% to 20.08%, with values of 9.44% (F1), 13.69% (F2), and 20.08% (F3) in female samples and 8.28% (M4), 19.77% (M5), and 8.47% (M6) in male samples. In addition to Bacillus, genera such as Ammoniphilus and Rubrobacter were also detected across the samples, along with several uncultured or unclassified genera present at lower relative abundance, reflecting the overall diversity of the rhizosphere microbial community.

Comparative abundance analysis between male and female rhizosphere

To further examine sex-associated differences in rhizosphere microbial composition, a comparative abundance analysis was conducted using mean relative abundance values of male and female samples. Relative-abundance differences between the groups were calculated using female abundance as the reference (Table 2). The analysis revealed that orders including Micromonosporales (50.98%), Thermomicrobiales (46.68%), Pedosphaerales (32.93%), and Chitinophagales (31.63%) showed comparatively higher mean relative abundance in female samples. At the phylum level, Bdellovibrionota (61.94%), Entotheonellaeota (61.29%), and Planctomycetota (49.68%) showed higher mean relative abundance in female rhizosphere samples. In contrast, certain bacterial groups belonging to Firmicutes and Bacteroidota, showed higher relative abundance in male rhizosphere samples. Despite differences in relative abundance, beta-diversity analysis showed no statistically significant separation between male and female microbial communities. Thus, no significant differences were observed in the overall microbiome composition between the two sexes, suggesting that sex-related differences in the date palm microbiome may not have a substantial influence on date palm productivity. However, the observed taxon-specific variation in relative abundance indicates that plant sex may still be associated with differences in specific microbial groups.

Heatmap analysis of dominant bacterial taxa

Heatmap analysis was performed to visualize the distribution and relative abundance patterns of dominant bacterial taxa across the rhizosphere samples (Figure 1). Variation in relative abundance patterns was observed across samples, particularly for members of Firmicutes, Proteobacteria, Actinobacteriota, and Acidobacteriota. Several dominant bacterial phyla were shared between male and female rhizosphere samples, although variation in relative abundance was observed. Firmicutes showed relatively greater abundance in male samples, whereas Proteobacteria and Actinobacteriota showed relatively greater abundance in female samples.

Beta diversity and microbial community structure

Beta diversity analysis was performed to assess differences in microbial community composition between male and female rhizosphere samples. Principal coordinate analysis (PCoA) based on weighted and unweighted UniFrac distance matrices was used to visualize patterns of similarity and dissimilarity among the samples. The PCoA plots (Supplementary Figures 14 and 15) based on weighted and unweighted UniFrac distance matrices showed partial overlap between male and female samples with respect to collection location, with no clear separation between groups. PERMANOVA indicated no statistically significant differences between male and female microbial communities for weighted UniFrac (pseudo-F = 0.79, R 2 = 0.157, p = 0.68; 999 permutations) and unweighted UniFrac (pseudo-F = 0.75, p = 0.91; 999 permutations). However, given the limited replication (n = 3 per sex), results should be interpreted cautiously as they may reflect low statistical power rather than true biological equivalence in community structure between sexes.

Differential abundance of microbial taxa (LEfSe analysis)

LEfSe analysis based on mean relative abundance identified bacterial taxa differentially enriched between male and female rhizosphere samples (Figure 2). Taxa meeting the predefined LDA effect-size threshold (≥2.0) and Kruskal–Wallis statistical significance threshold (p < 0.05) were considered discriminant. Male rhizosphere samples showed higher relative abundance of taxa including Bacteroidales, Rikenellaceae, Lactobacillales, Oscillospirales, Ruminococcaceae, and Desulfovibrionales. Female rhizosphere samples showed enrichment of Planctomycetota lineage, including Phycisphaerae, Tepidisphaerales, and Planctomycetales. Members of the WD2101 soil group and related candidate divisions also showed greater relative abundance in female samples. The complete list of discriminant taxa identified by LEfSe is provided in Supplementary Table 2.

Rhizosphere microbial diversity differences between male and female date palm plants

The present study revealed variation in rhizosphere microbial richness and diversity between male and female date palm plants. Female rhizosphere samples generally exhibited comparatively greater microbial richness and Shannon diversity than male samples, although differences in observed features were not statistically significant, and Shannon diversity showed only a borderline significant difference between groups. Similar sex-associated differences in rhizosphere microbial diversity have been reported in dioecious plant systems, where plant sex influences rhizosphere microbial communities through differences in physiology, metabolism, and root-associated processes (Zhou et al., 2022; Mao et al., 2024; Lan et al., 2025). The observed variation may reflect subtle differences in rhizosphere conditions associated with plant physiological status. Environmental influences and plant-associated factors can contribute to differences in microbial diversity patterns, which may not always result in statistically distinct microbial communities despite observable relative abundance variation (Li et al., 2024a; Wang et al., 2025). However, the observed differences in alpha diversity should be interpreted cautiously given the limited replication (n = 3 per sex).

Conserved taxonomic composition of the date palm rhizosphere microbiome

Despite relative abundance differences between male and female plants, the rhizosphere microbiome was broadly conserved across sexes and was dominated by Firmicutes, Proteobacteria, Actinobacteriota, Acidobacteriota, and Chloroflexi. These bacterial groups are commonly reported as dominant rhizosphere-associated taxa across diverse plant systems and represent major microbial components involved in rhizosphere functioning (Gao et al., 2025; Long et al., 2025).

At lower taxonomic levels, Bacilli, Bacillales, Bacillaceae, and Bacillus represented dominant components of the date palm rhizosphere microbiome. Similar hierarchical dominance patterns have been widely documented in rhizosphere microbial communities (Lv et al., 2025). The predominance of Bacilli together with proteobacterial classes is consistent with previous rhizosphere studies reporting these bacterial groups as dominant components of rhizosphere environments (Chen et al., 2026; Han et al., 2026). Their recognized roles in nutrient cycling and plant growth promotion further highlight their ecological importance in rhizosphere microbial dynamics and plant-microbe interactions (Tariq et al., 2025; Santoyo, 2025; Nicotra et al., 2026). Members of the genus Bacillus are recognized for their beneficial functions in rhizosphere environments, including nutrient mobilization, plant growth promotion, nutrient cycling, stress tolerance,and suppression of certain soil-borne pathogens (Du et al., 2025; Gayithri et al., 2026).

The predominance of Bacillus observed in the present study indicates that this genus is a prominent component of the date palm rhizosphere microbiome, although its functional contribution cannot be determined from the present 16S rRNA data alone. Despite dominant bacterial groups were shared between sexes, subtle differences in relative abundance were observed. Firmicutes showed a slightly higher mean relative abundance in male rhizosphere samples than in female samples, while Bacilli and related taxa also showed minor variation between the two sexes. These findings suggest that while core bacterial communities remain conserved, plant-associated factors may contribute to abundance-level variation within rhizosphere microbial communities. Similar patterns of diverse bacterial genera, including dominant taxa such as Bacillus, along with the presence of numerous uncultured microorganisms, have been widely reported in rhizosphere microbiome studies (Che et al., 2026; Jaramillo-Mazo et al., 2026).

Sex-associated variation in microbial relative abundance

Comparative analysis showed higher mean relative abundance of several bacterial taxa in female rhizosphere samples, including Bdellovibrionota, Entotheonellaeota, and Planctomycetota, together with bacterial orders such as Micromonosporales, Thermomicrobiales, Pedosphaerales, and Chitinophagales. Several of these taxa have previously been associated with processes such as organic matter turnover and nutrient cycling in soil environments (Pan et al., 2025) however, these functions were not directly assessed in the present study. Members of Micromonosporales have been associated with production of bioactive compounds and plant growth-promoting activities (Hifnawy et al., 2020; Gu et al., 2025), whereas Chitinophagales are recognized as rhizosphere-associated bacteria involved in degradation of complex organic substrates (Nhat et al., 2025). Similarly, Chloroflexi-related taxa, including Thermomicrobiales, were enriched in the studied soils, suggesting their potential involvement in soil metabolic processes (Guellout et al., 2026). Members of Verrucomicrobiota are often linked with soil carbon dynamics and participate in biogeochemical cycling through the degradation of complex organic compounds (Naziębło et al., 2026).

In contrast, certain bacterial groups, particularly those belonging to Firmicutes and Bacteroidota, showed relatively higher abundance in male rhizosphere samples. These groups are often associated with stress tolerance, rapid growth under fluctuating environmental conditions, and resilience in nutrient-limited soils (Haider et al., 2026), These differences represent taxon-specific relative-abundance variation between the two groups, while their functional significance remains to be experimentally determined. The observed differences in relative abundance indicate subtle variation in microbial composition between the sexes. Such patterns may be linked to plant physiological status, particularly increased nutrient demand during fruiting, which may influence root exudation and microbial recruitment. however, this possible mechanism requires further investigation (Li et al., 2024b).

Despite these relative abundance-based differences between male and female rhizosphere samples, assessment of overall microbial community structure revealed a contrasting pattern. Beta diversity analysis indicated no statistically significant separation between male and female rhizosphere microbial communities based on weighted and unweighted UniFrac analyses. Principal coordinate analysis similarly demonstrated substantial overlap between microbial communities across groups, indicating broadly conserved community composition. Despite similarity in overall community structure, exploratory LEfSe analysis and relative abundance-level variation revealed differential enrichment of specific bacterial taxa between male and female rhizospheres. Male rhizosphere samples showed enrichment of taxa belonging to Bacteroidales, Rikenellaceae, Lactobacillales, Oscillospirales, Ruminococcaceae, and Desulfovibrionales, which are commonly associated with organic matter degradation and fermentative processes, including the breakdown of complex polysaccharides such as cellulose, a major component of plant-derived organic matter (Pan et al., 2023; Yang et al., 2025).

In contrast, female rhizosphere samples were enriched with taxa belonging to the Planctomycetota lineage, including Phycisphaerae, Tepidisphaerales, and Planctomycetales, indicating consistent enrichment across multiple taxonomic levels. In addition, several uncultured and soil-associated bacterial lineages, including members of the WD2101 soil group and related candidate divisions, were also more abundant in female samples. Some of these taxa have previously been associated with soil nutrient cycling and organic matter turnover, suggesting a potential shift toward nutrient-transforming microbial communities in female rhizospheres. This enrichment may be associated with increased nutrient demand during the fruiting stage, which is consistent with previous studies demonstrating sex-dependent variation in rhizosphere microbial communities across floral and fruit developmental stages (Li et al., 2024b). This observation is further supported by studies highlighting the role of Planctomycetota and related soil bacterial groups in organic matter degradation and nutrient cycling in biomass-rich environments (Klimek et al., 2025). These observations indicate that while the overall community structure remains similar, differences in microbial relative abundance patterns may reflect subtle influences of plant physiological status on the rhizosphere microbiome. These observations are consistent with previous studies on dioecious plant systems (Cui et al., 2023; Lin et al., 2025). Such differences may have important implications for nutrient dynamics and soil ecosystem functioning, suggesting that plant sex can subtly influence rhizosphere processes through selective microbial recruitment, as demonstrated in recent studies on dioecious plants showing sex-dependent shifts in root-associated microbial communities and their functional potential across developmental stages and environmental conditions (Liu et al., 2024; Zhu et al., 2024).

Implications for sustainable rhizosphere management in date palm cultivation

The identification of microbial taxa associated with nutrient transformation and rhizosphere functioning highlights their potential application in microbiome-based strategies for sustainable date palm cultivation (Agorsor, 2026). Enriched bacterial groups, particularly those linked with nutrient cycling and plant growth promotion, may serve as promising candidates for the development of bioinoculants aimed at improving soil fertility, enhancing nutrient use efficiency, and supporting plant performance under arid and semi-arid conditions (Wang and Xu, 2026). Moreover, understanding sex-associated microbial recruitment patterns could enable more targeted rhizosphere management practices in dioecious cropping systems as plant reproductive traits can influence the assembly and composition of rhizosphere microbial communities (Liao et al., 2026). Future research should focus on larger sample sizes, individual plant-level replication, and the influence of plant variety, developmental stage, seasonal variation, environmental conditions, and sex-associated factors on rhizosphere microbiome composition. In addition, integrating functional approaches such as metagenomics, metatranscriptomics, and root exudate profiling will be essential to test the hypotheses regarding the ecological roles of the identified taxa and to translate these findings into practical applications for sustainable agriculture (Wang et al., 2026).

This study provides a comparative assessment of rhizosphere bacterial communities associated with male and female date palm plants using 16S rRNA gene amplicon sequencing. The findings indicate that the date palm rhizosphere microbiome is largely conserved across sexes, with both groups dominated by major bacterial phyla such as Firmicutes, Proteobacteria, and Actinobacteriota. Despite this overall similarity, exploratory differences in the relative abundance of specific taxa were observed, with female rhizospheres showing higher representation of groups including Bdellovibrionota (61.94%), Entotheonellaeota (61.29%) and Planctomycetota (49.68%) and orders such as Micromonosporales (50.98%), Thermomicrobiales (46.68%), Pedosphaerales (32.93%), and Chitinophagales (31.63%). These exploratory patterns suggest that plant sex may not strongly influence overall community structure but may be associated with differences in the relative abundance of microbial groups with potential functional relevance, reflecting differences in physiological status and resource demand. Collectively, these insights provide a foundation for exploring microbiome-assisted strategies to enhance soil health and crop performance in date palm cultivation. In a broader context, this study contributes to sustainable agriculture by supporting SDG 2 and SDG 12 and highlights the need for future research integrating larger sample sizes and functional approaches to better elucidate the ecological and practical significance of sex-associated microbial variation.