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.

A complete ecosystem is also a complex network in which mul-tifarious species interact with each other to achieve system-levelfunctions, such as nutrient biogeochemistry[1]. Microbial commu-nity is commonly considered as the primary driving force ofecosystem nutrient mobilization and metabolism, especially car-bon (C), nitrogen (N), phosphorus (P), sulfur (S) and methane cou-pling process[2]. The rise of metagenomics and high-throughputarray (e.g. PhyloChip, GeoChip, etc.) technologies enable acquiringthe detailed information on microbial community functional geneabundance and diversity[3,4]; however, there has been far lessattention focusing on the direct and indirect interactions betweennutrient cycling genes coexisting in environmental samples. Docu-menting these interactions between functional genes acrossdiverse microbial communities may help to clarify the functionalroles and even environmental niches in different contexts[5]. Withthe increasing accumulation of functional gene data from modernhigh-throughput technologies, we are facing the challenge of thoseinteraction explorations, and to extend analyses beyond sole abun-dance and diversity comparisons.

Recent evidence for intimate relationship of plants with their microbiota shows that plants host individual and diverse microbial communities that are essential for their survival. Understanding their relatedness using genome-based and high-throughput techniques remains a hot topic in microbiome research. Molecular analysis of the plant holobiont necessitates the application of specific sampling and preparatory steps that also consider sources of unwanted information, such as soil, co-amplified plant organelles, human DNA, and other contaminations. Here, we review state-of-the-art and present practical guidelines regarding experimental and computational aspects to be considered in molecular plant–microbiome studies. We discuss sequencing and “omics” techniques with a focus on the requirements needed to adapt these methods to individual research approaches. The choice of primers and sequence databases is of utmost importance for amplicon sequencing, while the assembly and binning of shotgun metagenomic sequences is crucial to obtain quality data. We discuss specific bioinformatic workflows to overcome the limitation of genome database resources and for covering large eukaryotic genomes such as fungi. In transcriptomics, it is necessary to account for the separation of host mRNA or dual-RNAseq data. Metaproteomics approaches provide a snapshot of the protein abundances within a plant tissue which requires the knowledge of complete and well-annotated plant genomes, as well as microbial genomes. Metabolomics offers a powerful tool to detect and quantify small molecules and molecular changes at the plant– bacteria interface if the necessary requirements with regard to (secondary) metabolite databases are considered. We highlight data integration and complementarity which should help to widen our understanding of the interactions among individual players of the plant holobiont in the future.