Microbial rrn copy number is associated with soil C: N ratio and pH under long-term fertilization

Soil microbial life-history strategies, as indicated by rRNA operon (rrn) copy numbers, strongly influence agroecosystem functioning. Long-term N fertilization causes strong and lasting changes in soil properties, yet its impact on microbial strategies remains largely unexplored. Using long-term field experiments across three agroecosystems, we consistently found that N fertilization strongly decreased soil C: N ratio and pH, further increasing the community-level rrn copy number, including both average rrn copy number and total 16S rRNA copy number. Soil C: N stoichiometry balanced by N supplement favored the growth of N-dependent copiotrophic species containing high rrn copy numbers (an average of 2.5) and increased their network connections, predominantly contributing to community-level rrn copy number increase. Decreased soil pH caused by N fertilization also favored the growth of some species whose abundances negatively correlated with pH, partially contributing to the community-level rrn copy number increase. By examining the genomes of two dominant species, we found that microorganisms with a higher rrn copy number specialIntscript e.g., Streptomyces scabiei, , possessed more genes related to C , N transport and metabolism. In contrast, the Mycobacterium simiae with a lower rrn copy number specialIntscript has more genes associated with secondary metabolite biosynthesis and lipid transport and metabolism. Our finding challenges the concept of microbial life-strategy regulation solely by nutrient avail- ability, highlighting the important contributions of soil stoichiometric balance and pH to microbial strategies in agro-ecosystems under long-term N inputs.