Carbon Utilization Profiles and Nanopore 16S Sequencing Reveal Functional Divergence Among Strawberry Rhizosphere Microbiomes Under Alternative Ground Covers vs. Polyethylene Mulch

Sustainability of strawberry production depends on replacing polyethylene mulch with systems that reduce waste. Polyethylene mulch alters soil ecosystems; we hypothesized it would support greater diversity and metabolic potential than living mulches. Sequencing provides insight into microbial composition; functional differences reveal roles in nutrient cycling and soil health.

Soils were collected February 2024 from strawberry plots managed with perennial white clover, buckwheat–pea cover, sorghum–pea cover at 112 and 224 kg ha⁻¹, or polyethylene mulch at Rodale Institute (Camarillo, CA). Microbial functional potential was assessed using Biolog EcoPlates. Substrate utilization data were analyzed in R using GLM with a logit link, and Tukey pairwise comparisons. Positive/negative overal counts were evaluated using chi-square. Full-length 16S rRNA amplicons were sequenced on Oxford Nanopore MinION with a Flongle flow cell, basecalled using Dorado v0.9.5 (fast), and classified using EPI2ME wf-16s (v1.5.0). Shannon diversity and Pielou evenness were compared using one-sided Welch’s t-tests in Excel; beta diversity was assessed in Ranacapa using PERMANOVA.

Substrate utilization was similar for overall counts (χ² = 5.63, df = 4, p = 0.228). GLM detected treatment effects (p = 0.004), with perennial clover and both sorghum–pea differing from buckwheat–pea communities. Polyethylene mulch differed from perennial clover (p = 0.0464) and both sorghum treatments (p = 0.0464–0.0465), whereas buckwheat–pea remained similar to polyethylene. Significant substrates: α-cyclodextrin, α-ketobutyric acid, β-hydroxy-glycyl-L-glutamic acid, D-xylose, γ-aminobutyric acid, and Tween 40.

Plastic supported highest Shannon diversity (2.12) and Pielou evenness (0.377). Diversity and evenness exceeded perennial clover (Shannon: p = 0.0112; Pielou: p = 0.0101), and evenness exceeded sorghum–pea communities (p = 0.0419). Plastic and buckwheat–pea were enriched in Pseudomonadota; Bacillota dominated perennial clover and sorghum–pea. Findings suggest buckwheat–pea covers reproduce taxonomic structure and functional capacity of plastic rhizosphere microbiomes.