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Applying organic fertilizers has been well documented to facilitate the dissemination of antibiotic resistance genes (ARGs) in soil ecosystems. However, the role of soil fauna in this process has been seldom addressed, which hampers our ability to predict the fate of and to manage the spread of ARGs. Here, using high-throughput quantitative polymerase chain reaction (HT-qPCR), we examined the effect of long-term (5-, 8-, and 10-year) fertilization treatments (control, inorganic fertilizers, and mixed fertilizers) on the transfer of ARGs between soil, nematodes, and earthworms. We found distinct fates for ARGs in the nematodes and earthworms, with the former having higher enriched levels of ARGs than the latter. Fertilization impacted the number and abundance of ARGs in soil, and fertilization duration altered the composition of ARGs. Shared ARGs among soil, nematodes, and earthworm guts supported by a fast expectation-maximization microbial source tracking analysis demonstrated the trophic transfer potential of ARGs through this short soil food chain. The transfer of ARGs was reduced by fertilization duration, which was mainly ascribed to the reduction of ARGs in the earthworm gut microbiota. This study identified the transfer of ARGs in the soil-nematode-earthworm food chain as a potential mechanism for a wider dissemination of ARGs in the soil ecosystem.

The earthworm gut bacterial community and enzyme activities play a key role in many soil ecosystem functions, but how the addition of different types of biochar (used to improve soil quality) regulates soil and earthworm gut bacterial communities and associated enzyme activities is scarcely understood. An experiment was conducted using soil microcosms and seven treatments: no biochar application as control, and two types of biochar (maize straw and cow dung biochar, hereafter SB and CB treatments, respectively) with three biochar application rates (20, 50, and 100 g/kg soil). Eight enzymatic activities related to C-, N- and P-cycling (used to compute a “multifunctionality” index) and the abundance and composition of bacterial communities were assessed in both the soil and earthworm guts. Our results show that earthworm gut content, characterized by lower bacterial abundance but higher enzymatic activities than soil, was strongly influenced by biochar addition. Increasing biochar application rates significantly increased the nutrient content, bacterial abundance, and multifunctionality in the earthworm guts. Owing to the difference in physicochemical properties between SB and CB, the increased gut multifunctionality was linked to the significantly modified gut bacterial community composition in CB treatments but not in SB treatments. Structural equation modeling further revealed that the changed gut multifunctionalities in both biochar treatments were mostly related to the soil and gut physicochemical properties, especially in CB treatments, while the impacts of gut microbial variables on soil multifunctionality were higher in the SB than CB treatments. These results demonstrate that the influence of biochar amendment on earthworm gut microbiome and multifunctionality depends on the type of biochar, which also regulates the contributions of earthworm gut biochemical processes to soil ecosystem functions.

Most ecosystem functions attributed to earthworms are mediated by their internal microbiomes, and these are sensitive to disturbances in the external environment. However, few studies have focused on the response of the earthworm gut microbiome to soil chronosequence. Here, we used 16S rRNA high-throughput sequencing and high-throughput quantitative PCR to investigate the variations in bacterial communities and functional gene abundance in earthworm (Lumbricina sp.) guts and upland soils under 700 years of cultivation. Our results indicated that 700 years of upland cultivation significantly shaped bacterial communities and increased functional traits of microbes in earthworm guts, which were more sensitive to cultivation age compared to the surrounding soils. The earthworm gut bacterial community changed rapidly over the first 300 years of cultivation and then changed slowly in the following centuries. Along with the cultivation age, we also observed that the earthworm gut microbiota was successive towards a copiotrophic strategy (e.g., Xanthobacteraceae, Nocardioidaceae, Hyphomicrobiaceae, and Bacillaceae) and higher potential functions (e.g., ureC, nirS, nosZ, phoD, and pqqC). Furthermore, canonical correspondence analysis further revealed that soil pH, C:N ratio, soil organic carbon, and total nitrogen were key abiotic drivers shaping earthworm gut bacterial communities. Taken together, this study reveals the succession of bacterial communities and potential functions in earthworm guts within 700 years of upland cultivation, which may provide a broader space for us to rationally exploit and utilize the interactions between soil and earthworm gut microbiotas to benefit the soil nutrient cycling process.

The positive roles of earthworms on soil functionality has been extensively documented. The capacity of the earthworm gut microbiota on decomposition and nutrient cycling under long-term fertilization in field conditions has rarely been studied. Here, we report the structural, taxonomic, and functional responses of Eisenia foetida and Pheretima guillelmi gut microbiota to different fertilization regimes and durations using 16S rRNA gene-based Illumina sequencing and high-throughput quantitative PCR techniques. Our results revealed that the core gut microbiota, especially the fermentative bacteria were mainly sourced from the soil, but strongly stimulated with species-specificity, potential benefits for the host and soil health. The functional compositions of gut microbiota were altered by fertilization with fertilization duration being more influential than fertilization regimes. Moreover, the combination of organic and inorganic fertilization with the longer duration resulted in a higher richness and connectivity in the gut microbiota, and also their functional potential related to carbon (C), nitrogen, and phosphorus cycling, particularly the labile C decomposition, denitrification, and phosphate mobilization. We also found that long-term inorganic fertilization increased the abundance of pathogenic bacteria in the P. guillelmi gut. This study demonstrates that understanding earthworm gut microbiota can provide insights into how agricultural practices can potentially alter soil ecosystem functions through the interactions between soil and earthworm gut microbiotas.

Increasing the bioavailability of immobilized phosphorus (P) in soil by phosphate-solubilizing bacteria (PSB) is an effective strategy for sustainable agronomic use of P and for mitigating the P cr...

Paddy soil as a major component of cropland, plays an important role in the global carbon (C) cycle and favors carbon sequestration especially in southern China. Soil microorganisms are central to the conversion of organic matter into SOC, yet the mechanisms underlying the paddy management at long time scales remain largely unknown, including microbial enzyme and functional potential kinetics, microbial growth and turnover. Here, using observations from a 2000-year-old paddy chronosequence since reclamation from tidal wetland at two different soil depths (0-20 cm and 20-50 cm) in the Yangtze River Delta, China, we show how paddy soil C sequestration is driven by the relationship between short-term responses in microbial physiology and long-term changes in biogeochemical soil properties. The samples were analyzed for nutrient pools, microbial biomass and growth, microbial activity and community composition, functional gene abundances, as well as microbially mediated nitrogen (N) cycling rate to determine how these microbial functionalities and processes affect microbial carbon use efficiency (CUE), an important indicator for microbial C sequestration. Across multiple time-scales ranging from decades to millennia, SOC in topsoil was increased by 65% during the first 50 years and reached the steady-state condition until 700-year, then was largely accumulated by 169% and 125% in 1000- and 2000-year, respectively, while C loss appeared in subsoil after 700 years of paddy cultivation. For topsoil and subsoil, microbial CUE reached to the highest values in 1000- and 700-year (0.46 and 0.36, respectively, while only 0.20 in the tidal wetland), along with microbial growth which both increased 5.2- and 3.3-fold in 1000-year, respectively. We found the similar increasing trends between microbial CUE and soil C:P and N:P ratios, the reduction of N limitation and functional potentials including N- and P-cycling, C degradation, C-fixation (acsA gene), microbial community homogenization and microbial biomass across soil chronosequence in topsoil. Moreover, the structural equation model revealed that with longer paddy management, the decline in soil pH had positive effects on microbial functional potentials and microbial biomass carbon. The enhanced functional potentials directly positively affected microbial growth, and thereby on microbial biomass carbon. Finally, the prolonged paddy cultivation increased SOC content via its direct positive effect and indirect positive influence on microbial biomass carbon. We conclude that longer paddy management captures the cumulative microbial anabolism on SOC sequestration in the plough layer, with the shifts in abiotic and biotic conditions towards increased nutrient availability and homogenous microbial community with higher functional potentials.

Animal bodies are colonized by many microorganisms which can provide indispensable services to their hosts. Although nematode gut microbiota has been extensively studied in recent years, the driving factors of gut microbiome of soil nematodes from a long-term fertilization field are unclear. Here, using 16S rRNA gene amplicon sequencing, we explored the nematode gut microbiota under different fertilization patterns (control, inorganic fertilizers and mixed fertilizers) and fertilization durations (5 y, 8 y and 10 y). Our results revealed that nematode gut microbiota was dominated by core bacterial taxa AF502208 (anaerobic bacteria), Enterobacter (plant litter decomposition) and Ancylobacter (organic matter decomposition and nitrogen cycling), significantly distinct from soil microbiome, and the assembly of that was a non-random process, which suggested host conditions contributed to maintaining the gut microbiota. Moreover, fertilization pattern had a greater influence on nematode gut microbiome than fertilization duration. Inorganic fertilization (5.19) significantly reduced the diversity of the nematode gut microbiota (6.68) shown by Shannon index (P

Microorganisms mobilize phosphorus (P) from soil and make it available for plants. However, the role of microbial activity in soil P dynamics especially among different P pools is poorly understood largely due to methodological limitations. In this study, we analyzed the oxygen isotope ratios in phosphate (δ18OP) of sequentially extracted inorganic P (Pi) pools (H2O-Pi, NaHCO3-Pi, NaOH-Pi, and HCl-Pi) in a long-term agricultural research field in Henan, China with different fertilization histories and coupled with soil enzyme activity and P-cycling bacteria gene abundance studies. Results showed the dominant enzymes were alkaline phosphatase (APase) and phosphodiesterase (PDE), and the functional genes for P-cycling were bpp, cphy, phoX and pqqC. After long-term P fertilization, the δ18OP values of H2O-Pi, NaHCO3-Pi and NaOH-Pi pools approached to or achieved equilibrium, suggesting that the externally applied P was actively mobilized and cycled by soil microorganisms and speciated into different P pools. Based on the extent of isotope excursion among different Pi pools, the equilibrium fractionation oriented the source signature in the NaHCO3-Pi and NaOH-Pi pools, and HCl-Pi pool could be derived from P fertilizer but through multistep reactions, and mixed with the HCl-Pi from rock weathering product, which constituted HCl-Pi pool in the non-P treatments. Overall, the long-term P fertilization especially the balanced fertilization with nitrogen (N), P and potassium (K) was found to be beneficial for extensive utilization of soil P with abundant biological uptake and cell-internal Pi cycling. Variation partitioning analysis (VPA) indicated that the expression of functional genes may be stimulated to mobilize soil P under specific P pools distribution in this alkaline environment. Overall, our findings provide new insights to understand the roles of microbial activities in soil P biogeochemistry that are useful for agricultural P sustainability.

Although the roles of earthworms and soil collembolans in the transport of microplastics have been studied previously, the effects of the soil biota at different trophic levels and interspecific relationships remain poorly understood. Here, we examine three soil microarthropod species to explore their effects on the transport of microplastics. The selected Folsomia candida and Hypoaspis aculeifer are extensively used model organisms, and Damaeus exspinosus is a common and abundant indigenous species in China. A model food chain (prey-collembolan and predator-mite) was structured to test the role of the predator-prey relationship in the transport of microplastics. Commercial Polyvinyl chloride (PVC) particles (Diameter: 80-250 μm) were selected as the test microplastics, because large amounts of PVC have persisted and accumulated in the environment. Synchronized soil microarthropods were held in plates for seven days to determine the movement of microplastics. The 5000 microplastic particles were carefully placed in the center of each plate prior to the introduction of the animals. Our results clearly show that all three microarthropod species moved and dispersed the microplastics in the plates. The 0.54%, 1.8% and 4.6% of the added microplastic particles were moved by collembolan, predatory mite and oribatid mite, respectively. Soil microarthropods (0.2 cm) transported microplastic particles up to 9 cm. The avoidance behavior was observed in the collembolans in respect of the microplastics. The predatory -prey relationship did promote the transport of microplastics in the plates, increasing transport by 40% compared with the effects of adding single species (P .05). Soil microarthropods commonly occur in surface soils (0-5 cm) and, due to their small body size, they can enter soil pores. Our results therefore suggest that the movement of microplastics by soil microarthropods may influence the exposure of other soil biota to microplastics and change the physical properties of soils.

Earthworms are crucial regulators of many soil ecosystem processes; however, the effects of fertilization regimes on earthworm gut microbiota remains poorly understood. Here, we investigated the effects of 10 years of fertilization on the gut microbiota of two different earthworm species, Metaphire californica and Amynthas phaselus, belonging to anecic and endogeic ecological groups, respectively. Five fertilization treatments were designed as follows: non-nitrogen fertilizer (CK), nitrogen fertilizer only (NPK), and the combined application of inorganic fertilizers and three different application rates of pig manure (NPKM1, NPKM2, NPKM3). We observed that, compared with the CK and NPK treatments, the organic manure treatments had strong effects on both earthworm gut microbial communities and the associated metabolic functions. The lower application rate of organic manure significantly increased the gut microbial diversity of both earthworm species, which may have further improved their gut microbial metabolic potential, especially for the metabolism of various carbon resources. With the increasing application of organic fertilizers, the abundance of several bacterial families, such as Comamonadaceae, Oxalobacteraceae, and Cytophagaceae, significantly increased in the gut of M. californica. These families have also been identified as the primary indicator taxa of high organic matter inputs and play a crucial role in regulating microbial community functionalities. However, a similar trend was not observed in the gut of A. phaselus, indicating that A. phaselus might be less sensitive to fertilization regimes because it lives deeper in the soil and has a long digestive tract compared with those of M. californica. Our results demonstrated that long-term organic amendment application significantly shapes earthworm gut microbial communities and results in greater decomposition potential of the earthworm gut. Furthermore, our study found that lower application rates of organic matter may be beneficial for earthworm gut microbial diversity and functional structures.

Little is known about the effects of temperature and drying–rewetting on soil phosphorus (P) fractions and microbial community composition in regard to different fertilizer sources. Soil P dynamics and microbial community properties were evaluated in a soil not fertilized or fertilized with KH2PO4 or swine manure at two temperatures (10 and 25 °C) and two soil water regimes (continuously moist and drying–rewetting cycles) in laboratory microcosm assays. The P source was the dominant factor determining the sizes of labile P fractions and microbial community properties. Manure fertilization increased the content of labile P, microbial biomass, alkaline phosphomonoesterase activity, and fatty acid contents, whereas KH2PO4 fertilization increased the content of labile inorganic P and microbial P. Water regimes, second to fertilization in importance, affected more labile P pools, microbial biomass, alkaline phosphomonoesterase activity, and fatty acid contents than temperature. Drying–rewetting cycles increased labile P pools, decreased microbial biomass and alkaline phosphomonoesterase activity, and shaped the composition of microbial communities towards those with greater percentages of unsaturated fatty acids, particularly at 25 °C in manure-fertilized soils. Microbial C and P dynamics responded differentially to drying–rewetting cycles in manure-fertilized soils but not in KH2PO4-fertilized soils, suggesting their decoupling because of P sources and water regimes. Phosphorus sources, temperature, and water regimes interactively affected the labile organic P pool in the middle of incubation. Overall, P sources and water availability had greater effects on P dynamics and microbial community properties than temperature.

Soil drying-rewetting can remarkably affect phosphorus (P) transformation, and thus, alter P distribution among P pools; however, little is known about the effect of the degree of drying before rewetting on labile P fractions and the bioavailability of organic P (Po). In this study, soils with distinct physico-chemical properties were allowed to desiccate to 5%, 10%, 20%, 30%, and 40% water holding capacity (WHC), and soils maintained at 50% WHC were used as controls, and the bicarbonate-extractable P and hydrolyzable Po fractions were analyzed after 5 h of rewetting. Bicarbonate-extractable Po accounted for 33.3–56.4% of extractable total P, and hydrolyzable Po constituted 34.4–79.7% of extractable Po. For extractable Po, 7.7–29.9% was labile monoester P, 6.5–14.9% was diester P, and 17.8–36.5% was phytate-like P. Bicarbonate-extractable inorganic P and Po contents were not affected by 20–40% WHC treatments, but increased by 26.3–48.1% and 5.7–52.9%, respectively, when the soils were dried to 5% WHC. Similarly, labile monoester, diester, and phytate-like P contents increased by 12.5–89.8%, 0–65.2%, and 24.6–65.6%, respectively, in 5% WHC soils. Pearson's correlation analyses showed that the relative increases in bicarbonate-extractable inorganic P and phytate-like P, following extreme drought, were positively correlated with soil organic carbon, oxalate-extractable aluminum (Al), and iron (Fe), suggesting regulatory roles of organic matter and Al/Fe oxides in P transformation during soil drying-rewetting. Taken together, our results suggest that extreme drought events before rainfall or irrigation facilitate an increase in the level of labile P, including considerable proportions of hydrolyzable Po fractions, potentially posing a substantial threat to water bodies in the context of climate change.

A Gram-stain-negative and rod-shaped bacterial strain, 12-OD1T, with rock phosphate solubilizing ability was isolated from agricultural soil in Hailun, Heilongjiang, PR China. The isolate was affiliated to the genus Massilia , based on 16S rRNA gene sequence alignments, having the highest similarities with Massilia putida 6 NM-7T (98.67 %), Massilia kyonggiensis TSA1T (98.28 %), and Massilia norwichensis NS9T (98.07 %), respectively. The DNA G+C content was 67.72 mol% and DNA–DNA hybridization showed low relatedness values (less than 47 %) between strain 12-OD1T and other phylogenetically related species of the genus Massilia . The predominant isoprenoid quinone was Q-8 and the polar lipid profile comprised diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major fatty acids were C17 : 0 cyclo (25.4 %), C16 : 0 (23.4 %) and summed feature 3 (C16 : 1ω7c and/or C16 : 1 ω6c) (22.5 %), which differentiates it from close relatives within the genus Massilia . Combined genetic, physiological and biochemical properties indicate that strain 12-OD1T is a novel species of the genus Massilia , for which the name Massilia phosphatilytica sp. nov., is proposed, with the type strain 12-OD1T (=CCTCC AB 2016251T=LMG 29956T=KCTC 52513T).

Livestock manure has gradually become an alternative fertilizer for maintaining soil fertility, whereas excessive application of manure leads to the release of phosphorus (P) and toxic metals that may cause complex environmental risks. To investigate the accumulation and migration of P within soil profiles, a mesocosm experiment was conducted to analyze the content and leaching of soil P, metals, and dissolved organic carbon after different fertilization treatments, including control (no fertilizer, CK), chemical fertilizer (CF), chemical fertilizer combined low (CF + LPM) and high (CF + HPM) rate of manure application. Results showed that a high rate of manure application significantly enhanced the accumulation of total soil P (by ~14%) and P availability (easily-available P, by ~24%; Olsen-P, by ~20%) in topsoil, and also increased the content of easily-available organic P (EA-Po) in both topsoil and subsoil compared to the CK treatment. The migration of dissolved inorganic and organic P (DIP and DOP) in leachate within soil profiles was strengthened by manure application. Moreover, significant positive correlations between P, metals, and dissolved organic carbon (DOC) in leachate indicated that downward co-migration occurred within the soil profiles, and also suggested that excessive manure application can intensify the risk of P loss by increasing the migration of manure-derived DOC. Overall, our findings provide insights into P accumulation and migration within soil profiles after excessive manure application, which is useful for predicting the potential risk of P and metal leaching from paddy soils.

Increasing the bioavailability of immobilized phosphorus (P) in soil by phosphate-solubilizing bacteria (PSB) is an effective strategy for sustainable agronomic use of P and for mitigating the P crisis. Here, D

The optimization of more sustainable fertilization practice to relieve phosphorus (P) resource scarcity and increase P fertilizer utilization, a better understanding of the regulatory roles of microbes in P mobilization is urgently required to reduce P input. The genes phoD and pqqC are responsible for regulating organic and inorganic P mobilization, respectively. Using high-throughput sequencing, the corresponding bacterial communities harbored by these genes were determined. We conducted a 4-year rice-rice-crop rotation to investigate the responses of phoD- and pqqC-harboring bacterial communities to the partial replacement of inorganic P fertilizer by organic manure with reduced P input. The results showed that a combination of organic and inorganic fertilization maintained high rice yield, and also produced a more complex and stable phosphate mobilizing bacterial community, which contributed to phosphatase activities more than their gene abundances in the model analysis. Compared with the conventional mineral fertilization, organic-inorganic fertilization with the reduced P input slightly increased pqqC gene abundance while significantly enhanced the abundance of phoD-harboring bacteria, especially the genera Bradyrhizobium and Methylobacterium known as potential organic P mineralizers which can maintain high rice production. Moreover, the increased pH was the most impactful factor for the phoD- and pqqC-harboring bacterial communities, by promoting microbial P turnover and greatly increasing bioavailable P pools (H2O-Pi and NaHCO3-Pi, NaOH-Pi) in this P-deficient paddy soil. Hence, our study demonstrated that the partial replacement of mineral P with organic manure could reshape the inorganic phosphate solubilizing and alkaline-phosphomonoesterase encoding bacterial communities towards more resilient and effective to the high P utilization and productivity over intense cultivation, providing insights into the potential of soil microbes in the efficient management of agricultural P fertilization.

The alternation of drying and rewetting events could dramatically affect the biological and structural properties of soil and consequently influence nutrient transformation. To examine whether organic amendments could improve the resistance and resilience of microbial function (extracellular enzyme activities), community composition (phospholipid fatty acids), and soil structure to drying-rewetting alternation, cropland soils with or without wheat-straw amendment were allowed to desiccate in a microcosm for two months, followed by moist incubation for five weeks, and continuously moist treatments were maintained at 50% water holding capacity during the entire period, as a control treatment. Straw amendment increased microbial biomass, extracellular enzyme activities, the relative abundance of fungal groups, dissolved organic carbon, and proportion of large macroaggregates (> 2000 μm), but decreased mineral nitrogen and available phosphorus. The drying-rewetting treatment increased microbial biomass carbon and β-glucosidase activities by 10% and 13% in straw-amended soils, respectively, but not in unamended soils, and decreased the urease and alkaline phosphomonoesterase activities by > 15% in unamended soils, but not in amended soils. The contents of fungi, actinomycetes, Pseudomonas spp., and Bacillus spp. decreased with drying, and more so with the subsequent rewetting, but recovered by the end of the experiment. The drying-rewetting treatment caused a decrease in the nitrate content in both soils (> 10%) and an increase in the macroaggregates of straw-amended soils (~ 8%). These results indicated that improved soil aggregation, as a result of straw amendment, protected microbial communities from drought stress and that nutrient acquisition promoted the post-rewetting colonization of heterotrophic communities characterized by hydrolase production, which consequently facilitated aggregate re-formation. Thus, straw amendment positively contributed to aggregate turnover and to both microbial and enzymatic responses to drying-rewetting events, which suggests that straw amendment is favorable to maintain soil function under conditions of increasing rainfall variability.

Nitrification inhibitors (NIs) 3,4-dimethylpyrazole phosphate (DMPP) and dicyandiamide (DCD) have been used extensively to improve nitrogen fertilizer utilization in farmland. However, their comparative effects on ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in agricultural soils are still unclear. Here, we compared the impacts of these two inhibitors on soil nitrification, AOA and AOB abundance as well as their community structure in a vegetable soil by using real-time PCR and terminal restriction fragment length polymorphism (T-RFLP). Our results showed that urea application significantly increased the net nitrification rates, but were significantly inhibited by both NIs, and the inhibitory effect of DMPP was significantly greater than that of DCD. AOB growth was more greatly inhibited by DMPP than by DCD, and the net nitrification rate was significantly related to AOB abundance, but not to AOA abundance. Application of urea and NIs to soil did not change the diversity of the AOA community, with the T-RFs remaining in proportions that were similar to control soils, while the community structure of AOB exhibited obvious shifts within all different treatments compared to the control. Phylogenetic analysis showed that all AOA sequences fell within group 1.1a and group 1.1b, and the AOB community consisted of Nitrosospira cluster 3, cluster 0, and unidentified species. These results suggest that DMPP exhibited a stronger inhibitory effect on nitrification than DCD by inhibiting AOB rather than AOA.