Fertilization influences the soil microbiome. However, little is known about the effects of long-term fertilization on soil microbial metabolic pathways. In this study, we investigated the soil microbiome composition and function and microbial participation in the N cycle according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) functional annotation of different genes in a metagenomic analysis after long-term fertilization. Fertilizer application significantly changed the soil C/N ratio. Chemical fertilizer (NPK) treatment decreased soil pH, and chemical fertilizer combined with straw (NPK+S0.5) treatment increased ammonium nitrogen (NH₄⁺-N) but decreased nitrate nitrogen (NO₃⁻-N). NPK, NPK+S0.5 and S0.5 applications did not change the soil microbiome composition or dominant phylum but changed the relative abundances of microbiome components. Moreover, fertilizer significantly influenced metabolic processes, cellular processes and single-organism processes. Compared with a no-fertilizer treatment (CK), the NPK treatment resulted in more differentially expressed gene (DEG) pathways than the NPK+S0.5 and S0.5 treatments, and these pathways significantly correlated with soil nitrate nitrogen (NO₃⁻-N), available phosphorus (AP) and the moisture content of soil (MC). KEGG analysis found that fertilizer application mainly affected the ribosome, photosynthesis and oxidative phosphorylation pathways. S0.5 and NPK+S0.5 increased microbial nitrogen fixation, and NPK and NPK+S0.5 decreased amoA and amoB and accelerated denitrification. Thus, organic fertilizer increased N fixation and nitrification, and inorganic N fertilizer accelerated denitrification. We found that the function of the soil microbiome under different fertilizer applications could be important for the rational application of fertilizer and for environmental and sustainable development.

Lead (Pb) is one of the nonessential and toxic metals that threaten the environment and human health. Medicago sativa L. is a legume with high salt tolerance and high biomass production. It is not only a globally important forage crop but is also an ideal plant for phytoremediation. However, the biological and molecular mechanisms that respond to heavy metals are still not well defined in M. sativa. In this study, de novo and strand-specific RNA-sequencing was performed to identify genes involved in the Pb stress response in M. sativa roots. A total of 415,350 unigenes were obtained from the assembled cDNA libraries, among which 5,416 were identified as significantly differentially expressed genes (DEGs) (false discovery rate < 0.005) between cDNA libraries from control and Pb-treated plants. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the DEGs showed they mainly clustered with terms associated with binding, transport, membranes, and the pathways related to signal and energy metabolism. Moreover, a number of candidate genes included antioxidant enzymes, metal transporters, and transcription factors involved in heavy metal response were upregulated under Pb stress. Quantitative real-time PCR(qRT-PCR) validation of the expression patterns of 10 randomly selected candidate DEGs were consistent with the transcriptome analysis results. Thus, this study offers new information towards the investigation of biological changes and molecular mechanisms related to Pb stress response in plants.

Antibiotic tolerance has become an increasingly serious crisis that has seriously threatened global public health. However, little is known about the exogenous factors that can trigger the development of antibiotic tolerance, both in vivo and in vitro. Herein, we found that the addition of citric acid, which is used in many fields, obviously weakened the bactericidal activity of antibiotics against various bacterial pathogens. This mechanistic study shows that citric acid activated the glyoxylate cycle by inhibiting ATP production in bacteria, reduced cell respiration levels, and inhibited the bacterial tricarboxylic acid cycle (TCA cycle). In addition, citric acid reduced the oxidative stress ability of bacteria, which led to an imbalance in the bacterial oxidation–antioxidant system. These effects together induced the bacteria to produce antibiotic tolerance. Surprisingly, the addition of succinic acid and xanthine could reverse the antibiotic tolerance induced by citric acid in vitro and in animal infection models. In conclusion, these findings provide new insights into the potential risks of citric acid usage and the relationship between antibiotic tolerance and bacterial metabolism.