16S rRNA:rDNA ratios and cell activity staining reveal consistent patterns of soil microbial activity

Microbial activity plays a major role in the processes that support life on Earth. Nevertheless, across diverse ecosystems many microbes are in a state of dormancy, characterized by strongly reduced metabolic rates. Of the methods used to assess microbial activity-dormancy dynamics, 16S rRNA: rDNA amplicons (“16S ratios”) and active cell staining with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) are two of the most common, yet each method has its own limitations. To better understand the applicability and potential complementarity of these two methods, we conducted two experiments investigating microbial activity in the rhizosphere. In the first experiment, we treated corn rhizosphere soil with common phytohormones to simulate plant-soil signaling during plant stress, and in the second experiment, we used bean exposed to drought or nutrient enrichment to more directly assess the impacts of plant stress on soil microbial activity. Overall, 16S ratios revealed numerous taxa with detectable RNA but no detectable DNA. However, overarching patterns in percent activity across treatments were unaffected by the method used to account for active taxa, or by the threshold 16S ratio used for taxa to be classified as active. 16S ratio distributions were highly similar across microbial phyla and were only weakly correlated with ribosomal operon number. Lastly, over relatively short time courses, 16S ratios are responsive earlier than CTC staining, a finding potentially related to the temporal sensitivity of activity changes detectable by the two methods. Our results suggest that 16S ratios and CTC staining provide robust and complementary estimates of bulk community activity.ImportanceAlthough the majority of microorganisms in natural ecosystems are dormant, relatively little is known about the dynamics of the active and dormant microbial pools through both space and time. The limited knowledge of microbial activity-dormancy dynamics is in part due to uncertainty in the methods currently used to quantify active taxa. Here, we directly compared two of the most common methods (16S ratios and active cell staining) for estimating microbial activity in rhizosphere soil, and found that they were largely in agreement in the overarching patterns, suggesting that either method is robust for assessing comparative activity dynamics. Thus, our results suggest that 16S ratios and active cell staining provide robust and complementary information for measuring and interpreting microbial activity-dormancy dynamics in soils. They also support that 16S rRNA:rDNA ratios have comparative value and offer a high-throughput, sequencing-based option for understanding relative changes in microbiome activity.