Community characterization of soil from the Central Deccan Plateau dry tropical deciduous forest (Ceddtrof) in central India using 16S rRNA gene amplicon sequencing

Soil from the Central Deccan Plateau dry tropical deciduous forest (Ceddtrof) in Ceddtrof, northeast Maharashtra, is rich in organic nutrients from the degradation of long leaves of trees, balanced water activity, ionic concentration, intense sunlight, high temperatures, and organic matter decomposing and nurturing the soil microbiome (1–4). Ceddtrof typically contains Actinomycetes (Streptomyces spp.) that produce secondary metabolites (5), such as antibiotics (6–8). The knowledge gained from studying these environments can provide insights into the existence of Candidatus taxa (9). A thick soil layer, approximately 12 cm in thickness, was collected five times in a 60-feet by 60-feet plot by the composite sampling method (Table 1).

The upper 2-cm humus layer containing organic matter was discarded, and the remaining 10-cm soil layer was saved. Soil was collected five times to make one composite sample for each site. Six composite samples were collected, labeled as KVRF01–KVRF06, and separately packed in Nasco Whirl-Pak sampling bags (PW390, HiMedia Laboratories) (11, 12). All the samples were subsequently transported to the laboratory on dry ice. The environmental temperature and pH of the samples recorded at the sampling site were ~23°C and 6.5–7.5, respectively. The maximum temperature recorded during transportation was 5°C. DNA was extracted from the composite soil samples using a QIAGEN DNeasy PowerSoil Pro Kit 50 Prep (Cat. No. 47014) according to the manufacturer’s protocol (13). Locus-specific sequence primers (forward 5′GTGCCAGCMGCCGCGGTAA3′) and (reverse 5′GGACTACHVGGGTWTCTAAT3′), were used to amplify the V4 region of the bacterial 16S rRNA gene (14, 15). PCR amplicon libraries were prepared using Nextera XT DNA Library preparation kit (Illumina, Inc., USA) following the manufacturer’s instructions (15, 16). To obtain final libraries, final clean-up was conducted using AMPure XP beads, which were then examined for fragment size distribution using TapeStation (5067–5582, Agilent Technologies) and quantified using Qubit DNA (Q32854, Thermo Fisher Scientific) prior to sequencing. We used the Illumina MiSeq platform with paired-end 2 × 250 bp chemistry to sequence the 16S rRNA gene amplicon libraries. Taxonomic profiling was performed for feature, statistical analysis, differential abundance, and diversity analysis using the stand-alone QIIME2 version 2023.9 (10). A total of 7,207 operational taxonomic units were identified and belonged to 17 phyla (Table 1), such as Proteobacteria, Actinobacteria, Acidobacteria, Plactomycetes, Chloroflexi, Bacteroidetes, Verrucomicrobia, Gemmatimonadetes, Firmicutes, Crenarchaeota, Nitrospirae, Armatimonadetes, Elusimicrobia, Cyanobacteria, Chlamydiae, Chlorobi, and some other phyla, such as Parvachaeota and Tenericutes, Euryarchaeota, Fibrobacteres, Calditrix, and Spirochaetes were also detected. The remaining taxa <3% belonged to more rare taxa such as WS3, OP3, OD1, TM6, AD3, TM7, BRC1, FBP, OP11, GOUTA4, GN04, FCPU426, NKB19, WS2, GAL15, WPS-2, SBR1093, GN02, MVP-21, and SR1 (Fig. 1).

B.N.R. is thankful to the DBT-National Centre for Cell Science, Pune, for providing all necessary laboratory space, resources, institutional funding for research, and a sequencing facility and University Grants Commission, India, for funding support (grant ID: PDFSS-2013–14-ST-MAH-4350).