Culture-Dependent and Culture-Independent Sequencing Reveal Representative Rhizosphere Bacterial Communities under Cover Crop and Plastic Film Mulch Management Systems

Long-term soil management practices influence microbial metabolism and nutrient cycling. This study evaluated the complementary use of culture-dependent Sanger sequencing and culture-independent Oxford Nanopore sequencing to characterize bacterial communities associated with zucchini (Cucurbita pepo), bell pepper (Capsicum annuum), and eggplant (Solanum melongena) following three years of contrasting soil management at the Rodale Institute for Organic Agriculture (Camarillo, California, USA). Field plots were managed using conventional plastic film mulch or a crimped winter cover crop consisting of pea (Pisum sativum) and Merced rye (Secale cereale cv. Merced). Rhizosphere soils were collected at the conclusion of the third growing season (October 2025). Community DNA was extracted using the Qiagen PowerSoil Pro Kit and sequenced on an Oxford Nanopore MinION. In parallel, cultivable rhizosphere bacteria were isolated using standard microbiological techniques, characterized by Gram staining, catalase testing, and MacConkey agar, and identified by 16S rRNA gene Sanger sequencing following Chelex® DNA extraction, PCR amplification, agarose gel verification, BLAST analysis, and Neighbor-Joining phylogenetic reconstruction on DNA Subway 2.0.

Sanger sequencing identified cultured isolates dominated by Bacillus, Priestia, Rossellomorea, Pseudomonas, Ensifer, and other rhizosphere-associated genera. Culture-dependent Sanger sequencing and culture-independent Nanopore sequencing showed strong qualitative concordance, with nine of the eleven cultured genera—including Bacillus, Priestia, Rossellomorea, Pseudomonas, Ensifer, Rhizobium, Agrobacterium, Lysobacter, and Paenibacillus—also detected in the metagenomic dataset. While Sanger sequencing enabled phenotypic characterization, phylogenetic analysis, and isolate-level identification of cultivable bacteria, Nanopore sequencing captured a broader spectrum of the rhizosphere microbiome, including uncultured and low-abundance taxa that were not recovered by cultivation. Together, these complementary approaches provided concordant yet distinct perspectives on the soil bacterial community, demonstrating that cultured isolates represented dominant members of the broader microbiome while metagenomic sequencing expanded community characterization.