Organic phosphorus (Po) may play a vital role in phosphorus availability via its mineralization by alkaline phosphatase (ALP), being encoded by phoD gene, in calcareous soil. Understanding the effects of long-term fertilization on the community of phoD harboring bacteria and the related alteration of the P availability owing to the changes in ALP secretion may offer a chance to elucidate the Po contribution to soil available P. Based on a long-term experiment, we analyzed the phoD gene harboring microbial diversity, abundance and composition, ALP and Po forms, and their relationship. The treatments involved were control without any fertilizers (CK), synthetic nitrogen and potassium (NK), synthetic nitrogen, phosphorus and potassium (NPK), NPK and crop stalk return (SNPK), and NPK plus organic manure (MNPK). Fertilization increased the abundance and diversity of phoD gene harboring microbial over CK. Those receiving NPK and NPK treatments integrated with organic supplements significantly improved the relative abundance of Proteobacteria but decreased Gemmatimonadetes at the phylum level, while all fertilized treatments appreciably increased the relative abundance of Lysobacter but decreased that of Gemmatirosa and Afipia, at the genus level. SNPK and MNPK treatments noticeably increased the relative abundance of Methylobacter but reduced Pseudomonas and Streptomyces relative to those receiving synthetic fertilizer treatments. Long-term fertilization markedly raised ALP activity, which was significantly and positively correlated with the relative abundance of the phylum Proteobacteria as represented by the genera Methylobacterium and Lysobacter. ALP was closely associated with moderately labile Po, followed by enzyme P, recalcitrant Po, and labile Po. The changes in phoD bacteria and ALP were mainly driven by soil organic carbon, Olsen P and pH. We concluded that the long-term fertilization, especially the addition of organic supplements, profoundly modified the soil properties and subsequently changed the diversity and relative abundance of phoD gene harboring bacteria, which promoted the activity of ALP, and thus the mineralization of various forms of Po (mainly moderately labile Po) to enhance the P availability.

Recording and tracking the long-term dynamic changes in microbial populations is as essential as monitoring other soil properties for evaluating soil quality and health; however, this area has significantly lagged due to technical constraints and challenges in storing fresh soil samples. Historically archived soil samples offer a unique opportunity to characterize the temporal dynamics of microorganisms over several decades. To determine whether archived air-dried soils can be utilized for this purpose, we compared the structure and composition of bacterial communities across fresh soils, air-dried soil archives stored for varying durations, and their corresponding rewetted counterparts, all sourced from a long-term fertilization experiment on calcareous loess soil. Soil microbial features were characterized using the MiSeq sequencing platform. The results indicated that the similarity of DNA-based bacterial community composition between fresh soil and both archived and rewetted soils followed a downward quadratic curve as archiving time increased. Specifically, the DNA-based community structure of soils air-dried and preserved for one year, as well as those rewetted after eight years of archiving, remained highly similar to that of fresh soil. Regarding taxonomic shifts, the relative abundance of Actinobacteria in both air-dried and rewetted soils increased with storage time. Conversely, the relative abundances of Acidobacteria and Gemmatimonadetes significantly increased in air-dried soils but decreased upon rewetting over time. The relative abundances of Chloroflexi and Firmicutes remained stable in air-dried soils; however, after rewetting, the former decreased while the latter increased dramatically. Furthermore, Proteobacteria, Rokubacteria, Planctomycetes, Bacteroidetes, and Latescibacteria exhibited a decreasing trend in both air-dried and rewetted soils. These findings suggest that air-dried soils preserve DNA-based community profiles more effectively than rewetted soils, particularly for samples stored for less than eight years. This study provides a valuable reference for utilizing archived historical soil samples from long-term experiments to investigate microbial community evolution.