Effects of a bacterial consortium and organic compost amendment on spinach (Spinacia oleracea L.) rhizosphere and endosphere microbiomes using 16S rRNA amplicon sequencing.

This study assesses the impact of a six-member bacterial consortium, applied in combination with an optimized organic compost amendment (OCM), on the growth and nutrient uptake of spinach (Spinacia oleracea L.) cultivated in nutrient-poor sandy loam soil in Purulia, West Bengal, India. The primary goal is to evaluate a sustainable biofertilizer strategy combining water hyacinth compost, chicken manure, and native microbial isolates to enhance plant biometric parameters, chlorophyll content, and iron accumulation. The study compares unamended control soil (CAM) against soil treated with the optimized organic amendment (OCM), soil treated with amendment plus the microbial consortium (PR), and spinach leaf endophytes (BP).We employed 16S rRNA gene amplicon sequencing to profile microbial community structures. DNA was extracted using the DNeasy PowerSoil Kit (Qiagen). The V3-V4 regions were amplified for soil samples, while the V5-V7 regions were amplified for endophytic bacterial communities. The bacterial consortium used as inoculants comprised MP1 = Pseudomonas sp.; MP2 = Azospirillum sp.; MP3 = Bacillus sp.; MP4 = Beijerinckia sp.; and MP5 = Flavobacterium ummariense, which were identified by 16S rRNA gene sequencing using the V3-V4 region. Sequencing was performed on the Illumina platform. Raw reads were processed in QIIME 2 (v1.7.0) using DADA2 for denoising. Taxonomy was assigned via a naive Bayes classifier against the Greengenes database. Statistical analyses of alpha and beta diversity were conducted in R (v4.4.0) using phyloseq (v1.50.0) and vegan (v2.7-2) packages.Sequencing yielded high-quality data (Q30 > 93 percent). Alpha diversity analysis revealed that OCM and PR treatments significantly increased microbial richness compared to the control (CAM), with PR exhibiting peak richness (Observed species 3010; Chao1 3041) and phylogenetic diversity. In contrast, the endophytic niche (BP) displayed low diversity, typical of selective colonization. Beta diversity analysis (Bray-Curtis) indicated near-total community restructuring between CAM and OCM treatments (dissimilarity 0.9877). PR showed very high ecological stability, characterized by high species turnover (beta_sim = 0.6221) and low nestedness (beta_sne = 0.0657).Taxonomic profiling revealed distinct dominant patterns. The control soil (CAM) was dominated by Bacillus mannanilyticus and Lysinibacillus sp. The PR treatment resulted in the most even community structure (Pielou 0.76; Berger-Parker 0.15), dominated by Bacillus aryabhattai, Microbispora rosea, Pseudomonas alcaligenes, and Paenibacillus. The endophytic community (BP) was highly dominated (more than 90 percent) by Flavobacterium ummariense. Rank reversal analysis confirmed that taxa such as B. aryabhattai and M. rosea gained significant dominance in PR, validating their selection for the consortium.Spatial aggregation indices (VMR, Morisita) revealed that PR supported the most uniform bacterial distribution, indicated by the lowest VMR (147.894) and Morisita index (6.360), whereas endophytes were highly clustered, showing extreme aggregation (BP VMR 27223.158; Morisita 20.680). Functional prediction (Tax4Fun and KEGG) indicated that the PR community was enriched in genes related to nutrient cycling (nitrogen and nickel transport), fatty acid synthesis, and root colonization. The endophytic community (BP) showed high enrichment in iron receptor proteins (K02014) and transposases. The data correlate these specific microbiome shifts with a 216 percent increase in leaf area, a 7.5-fold increase in total chlorophyll, and a 73 percent increase in leaf iron concentration.Footnote:During sequencing and data processing, samples were coded as CAM (control soil) and PR (OCM plus microbial inoculants). For manuscript consistency, these treatments are referred to as CP and OCM+MI, respectively. Accordingly, CAM = CP and PR = OCM+MI.