Variations of earthworm gut bacterial community composition and metabolic functions in coastal upland soil along a 700-year reclamation chronosequence.

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.

How can fertilization regimes and durations shape earthworm gut microbiota in a long-term field experiment?

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.

Fates of Antibiotic Resistance Genes in the Gut Microbiome from Different Soil Fauna under Long-Term Fertilization.

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 driving factors of nematode gut microbiota under long-term fertilization.

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 < 0.05). Canonical correspondence analysis demonstrates that soil properties such as pH, organic matter, total phosphorus, available phosphorus, ammonium nitrogen, moisture content, nitrate nitrogen and total nitrogen have significant effects on the nematode microbiome. Structured equation models further revealed that fertilization could obviously affect the nematode gut microbiota, and the effects were maintained even when accounting simultaneously for the drivers of soil bacteria and soil properties. This study provides a solid evidence that the shifting of nematode gut microbiota under long-term fertilization was resulted from environmental factors and host conditions, and advance the insights into host-microbiome in the agricultural ecosystems.

Partial replacement of inorganic phosphorus (P) by organic manure reshapes phosphate mobilizing bacterial community and promotes P bioavailability in a paddy soil.

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.

Increased risk of phosphorus and metal leaching from paddy soils after excessive manure application: Insights from a mesocosm study.

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.

D2O-Isotope-Labeling Approach to Probing Phosphate-Solubilizing Bacteria in Complex Soil Communities by Single-Cell Raman Spectroscopy.

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, D2O isotope labeling combined with single-cell Raman spectroscopy (Raman-D2O) was developed as an efficient activity-based approach to characterizing the presence and activity of PSB in a culture-independent way. On the basis of the finding that PSB were significantly more active than non-PSB in the presence of insoluble P, a C-D Raman band from active assimilation of D2O-derived D was established as a biomarker for both inorganic-phosphate-solubilizing bacteria and organic-phosphate-solubilizing bacteria. C-D ratios (intensities of C-D bands as percentages of the intensities of both the C-D and C-H bands) were further established as semiquantitative indicators of P-releasing activities because of the consistency between the C-D ratio and the concentration of solubilized phosphate or acid phosphatase activity as measured by conventional bulk assays. By applying Raman imaging, single-cell Raman-D2O clearly discerned PSB in a mixed-soil bacterial culture and even in complex soil communities. Remarkable heterogeneity of microbial activity, ranging from 2 to 30% (close to that in medium without P and that in medium with sufficient soluble P, respectively), was revealed at the single-cell level and clearly illustrated the subpopulation of soil bacteria active in solubilizing P. This work not only enables probing PSB and their P-releasing activities but also opens a window to explore more diverse microbial resources when obtaining related isotope-labeled substrates is prohibitive.

Identification and characterization of inorganic-phosphate-solubilizing bacteria from agricultural fields with a rapid isolation method.

The ability to solubilize fixed inorganic phosphorus (P) for plant growth is important for increasing crop yield. More P can be released by inoculating soil with inorganic-phosphate-solubilizing bacteria (iPSBs). We used 96-well microplates instead of traditional 200-mm petri dishes to rapidly screen iPSB strains for their solubilizing ability. We simultaneously obtained 76 iPSB isolates from 576 wells containing two agricultural soils. This method conveniently identified positive iPSB strains and effectively prevented fungal cross-contamination. Maximum-likelihood phylogenetic trees of the isolated strains showed that Bacillus megaterium was the most dominant iPSB, and strains Y99, Y95, Y924 and Y1412 were selected as representatives for the analysis of P solubilization. Succinic acid was the main organic acid of B. megaterium for releasing P. It was strongly correlated with the increase in soluble P concentration during 168 h of incubation of these four strains. pH was negatively exponentially correlated with the amount of soluble P in the medium, and the amount of succinic acid was strongly linearly correlated with the amount of P released (P < 0.001), suggesting that organic acid may mobilize microbial P. Our study provides an efficient and effective method for identifying and analyzing the growth of iPSB strains able to solubilize inorganic P and gives a better understanding of the mechanism of P solubilization.

Trophic predator-prey relationships promote transport of microplastics compared with the single Hypoaspis aculeifer and Folsomia candida.

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.

Effects of combined application of nitrogen fertilizer and biochar on the nitrification and ammonia oxidizers in an intensive vegetable soil.

Soil amended with single biochar or nitrogen (N) fertilizer has frequently been reported to alter soil nitrification process due to its impact on soil properties. However, little is known about the dynamic response of nitrification and ammonia-oxidizers to the combined application of biochar and N fertilizer in intensive vegetable soil. In this study, an incubation experiment was designed to evaluate the effects of biochar and N fertilizer application on soil nitrification, abundance and community shifts of ammonia-oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) in Hangzhou greenhouse vegetable soil. Results showed that single application of biochar had no significant effect on soil net nitrification rates and ammonia-oxidizers. Conversely, the application of only N fertilizer and N fertilizer + biochar significantly increased net nitrification rate and the abundance of AOB rather than AOA, and only AOB abundance was significantly correlated with soil net nitrification rate. Moreover, the combined application of N fertilizer and biochar had greater effect on AOB communities than that of the only N fertilizers, and the relative abundance of 156 bp T-RF (Nitrosospira cluster 3c) decreased but 60 bp T-RF (Nitrosospira cluster 3a and cluster 0) increased to become a single predominant group. Phylogenetic analysis indicated that all the AOB sequences were grouped into Nitrosospira cluster, and most of AOA sequences were clustered within group 1.1b. We concluded that soil nitrification was stimulated by the combined application of N fertilizer and biochar via enhancing the abundance and shifting the community composition of AOB rather than AOA in intensive vegetable soil.

Massilia phosphatilytica sp. nov., a phosphate solubilizing bacteria isolated from a long-term fertilized soil.

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 putida6 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).