Effects of tobacco stalk biochar application frequency on plant growth and soil quality: Evidences from metagenomics and pseudo-targeted metabolomics approaches

With the improvement of technologies for the resource utilization of agricultural wastes, tobacco stalk biochar is gradually being applied to agricultural production in karst regions. However, the effects of tobacco stalk biochar application at different frequencies on soil quality and plant growth are poorly understood. Here, we integrated metagenomics with soil pseudo-targeted metabolomics to explore how tobacco stalk biochar application frequencies affect soil chemical properties, enzyme activities, microbial communities, metabolites, and plant growth. The results showed that annual application of an equal amount of tobacco stalk biochar could better promote tobacco growth by increasing soil available nutrients and enzyme activities. Microbial community analysis revealed that annual application recruited beneficial bacterial genera ( Sphingomonas , Actinomadura and Variovorax ), while biennial application enriched microbial diversity. Moreover, soil pseudo-targeted metabolomics analysis showed that the annual application significantly increased the amino acid content, while one-time application significantly increased the phenolic acid. Additionally, redundancy analysis revealed alkali-hydrolyzable nitrogen (AHN) and pH as primary environmental factors synergistically regulated soil enzymes, microbial communities, and soil metabolites, thereby forming a complex interaction network. Phenylalanine, glutamate, pyroglutamic acid, polyphenol oxidase, Bradyrhizobium , and Pseudolabrys are likely key factors in network for mediating the effects of tobacco stalk biochar application frequency. Our results indicated that soil enzymes, microbial communities, and metabolites collectively drove the optimal enhancement of soil quality and plant growth under the annual equal application, suggesting a potential ways to mitigate the negative effects in continuous cropping obstacles. • Metagenomics and soil pseudo-targeted metabolomics were explored to assess soil improvement with biochar. • Alkali-hydrolyzable nitrogen and pH serve as primary environmental drivers. • Six key factors respond to biochar application frequency. • Annual application of tobacco stalk biochar achieves the most effective improvement for karst yellow earth and plant growth.