Carbon And Nitrogen Transformations And Microbial Diversity In The Rhizosphere Soil Under Long-term Fertilization Practices

Carbon and nitrogen cycling in the rhizosphere are very important for soil nutrient transformations and plant nutrient availability. Mechanisms of microbes-mediated carbon and nitrogen cycling in the rhizosphere can be revealed by a long-term fertilizer field experiment, which can give a more reliable result compared with short-term experiment. In this study, extracellular enzyme activity, microbial community structure, microbial utilization pattern of root-derived C and microbial ammonia oxidation and denitrification processes in the rhizosphere(wheat and maize) soil from a long-term(36-year) fertilizer field experiment were investigated by using DGGE, PLFA, SIP-DNA, quantitative PCR, 454-pyrosequencing and Illumina Mi Seq Platform. The main findings obtained are as follows:1. Crop yield and rhizosphere nutrient status under long-term fertilization practices. Except for NO3--N, nutrient concentrations were generally higher in the rhizosphere than in the bulk soil. Both inorganic or organic fertilizer amendments increased wheat yield by 344-521%, and increased maize yield by 63-68%. However, organic manure was more conducive to the improvement of soil fertility compared with inorganic fertilizer, because organic manure not only can promote crop growth, but also enhance soil organic matter and total N.2. Rhizosphere microbial biomass and enzyme activity under long-term fertilization practices. Soil microbial biomass(SMB)-C,-N and-P in wheat rhizosphere were 1.9-fold, 2.5-fold and 1.8-fold greater than those in bulk soil. However, lower ratio of SMB-C to SMB-N was observed in the rhizosphere compared with bulk soil, indicating that more N was immobilized directly in rhizosphere microorganisms which became a potential N source for crops. Inorganic fertilizers did not significantly affect microbial biomass in the rhizosphere, whereas organic manure increased SMB-N and SMB-P by 85.8-178.3% compared with CK. There was a profound rhizosphere effect on soil enzyme activity, as indicated by an average increase in 11 extracellular enzyme activities of 87% in the rhizosphere when compared with bulk soil. In the bulk soil, both inorganic and organic fertilizations signi?cantly increased the activities of α-glucosidase, β-xylosidase, β-cellobiosidase, N-Acetyl-glucosaminidase, β-glucosidase, Phosphatase and urease. In the rhizosphere, however, inorganic fertilizers generally maintained or reduced these enzyme activities.3. Rhizosphere microbial community structure under long-term fertilization practices. Higher total PLFA(averagely increased by 80%) and lower ratios of G+ to G-, of bacteria to fungi and of actinomycetes to fungi were observed in the rhizosphere compared with the bulk soil. Long-term application of organic manure significantly increased microbial biomass. Total PLFAs in manure-treated soils were 1.7-2.0 times of those in CK-treated soils. Meanwhile, Microbial community structure in manure-treated soils was markedly different from those in inorganic fertilizer-treated soils. However, in the rhizosphere there were no statistically signi?cant differences in the abundance of bacteria, fungi and actinomycetes between the inorganic and organic treatments. Moreover, organic C, inorganic N and soil p H were important factors, which markedly affected microbial community structure. DGGE analysis showed that Proteobacteria was the dominant phylum in the ?uvo-aquic soil, and its abundance was increased in organic treatment.4. Microbial decomposition of rhizodeposits under long-term fertilization practices. Wheat root-derived 13 C was dominantly assimilated by Actinobacteria and Proteobacteria, accounting for nearly 70% of root-feeding microbiome. In contrast, Acidobacteria, Chloro?exi, Firmicutes and Bacteroidetes mainly utilized original SOM as their main C source in the wheat rhizosphere. Long-term application of organic manure facilitates the development of microbial communities with high biodiversity, whereas inorganic fertilizers selectively activate speci?c populations such as Actinobacteria and Nitrospirae, leading to a lower soil microbial diversity.5. Rhizosphere ammonia oxidation under long-term fertilization practices. The rhizosphere PNA ranged from 1.5 to 3.1 μg NO3--N g-1 h-1, and was about 42.7% higher than that in bulk soil. Our results indicate that AOB rather than AOA functionally dominate ammonia oxidation in the calcareous fluvo-aquic soil, even though the abundance of AOA was 10-fold greater than that of AOB. Long-term fertilization significantly affected the community structure of AOB, resulting in more abundant AOB phylotypes similar to Nitrosospira clusters 3 and 4 in the N-fertilized treatments, whereas organic manure significantly enhanced the AOA population size by 113.2%.6. Rhizosphere denitrification under long-term fertilization practices. There was an unaccounted yet abundant denitrifyingbacterial community within wheat root. The abundance of nir K, nir S or nos Z genes in root-endophytic community was both greater than 1.0×109 copies g-1 dw root. N2O/(N2O+N2) of wheat roots were significantly higher than those of the rhizosphere soil. The Mi Seq sequencing result showed that nir K, nir S and nos Z-type denitrifiers communities within wheat root had lower species diversity compared with rhizosphere soil, and were dominanted by Rhizobiales, Rhodobacterales, Burkholderiales, Pseudomonadales, Xanthomonadales(accounting for nearly 79% of endophytic denitrifiers). Both inorganic and organic fertilizer amendments significantly decreased the ratio of nir K to nir S gene, and increased the ratio of N2 O to N2O+N2 within wheat root.In summary, long-term applications of inorganic fertilizers increased the abundances of Actinobacteria and Nitrospirae in the rhizosphere soil, especially for AOB, accelerated the transformation of NH4+to NO3-, but decreased soil microbial diversity and rhizosphere enzyme acticities. In contrast, long-term application of manure significantly increased the abundances of Chloro?exi, Bacteroidetes, Firmicutes, AOA and denitrifiers. The alterations of the soil bacterial community composition caused by the application of inorganic fertilizers could be resumed through the addition of organic manure.