Nitrogen Dynamics And Microbial Diversity In Soils Under Organic Vegetable Production

Even though nitrogen(N) is among the most abundant elements on earth, it is also the major element limiting growth of plants in many agricultural systems because of its unavailability for plants. In low input systems crop growth is substantially limited by the exclusive and insufficient N supply from organic matter mineralization and crop yields can be reduce considerably compared with conventional systems. In light of the critical role N plays for agricultural production and its limitation for crop growth in low input systems, understanding the controls over N dynamics is central to management practice. Due to the widespread ability of plants to uptake and assimilate organic N and its potential as important N source, in the recent years organic N pools have been quantified in soil of several forest ecosystems, however we know little about this pool in agricultural systems. The microbial community and microbial processes play a critical role in nutrient biogeochemical cycles, such as in organic matter decomposition and nutrient cycling carried out by bacteria and fungi. Atmospheric N fixation, carried out with the aid of the nitrogenase enzyme manufactured by some prokaryotic microorganisms, is widely recognized as an important process in many ecosystems and can act as an important source of N in low input systems. Therefore, gaining an understanding of the ecology of the soil microbiota involved in N transformations and how their distribution and abundance are affected by farming systems and soil environmental properties can be used to better understand the biogeochemical cycling of N in agroecosystems and for sustainable soil management.In these series of experiments we investigated, using the nif H gene, a marker gene for the microbial community involved in N fixation, the occurrence(DNA) and activity(RNA) of the diazotrophic community in organically and conventionally managed soils in a horticultural system over the course of one year. For that, a soil field study was designed in which each management-cultivation method type was sampled in three replicated fields along four seasons(i.e., May, July and November, 2010 and February, 2011). The changes in the diazotrophic community structure were analyzed by PCR-denaturing gradient gel electrophoresis(DGGE). The abundance of the nif H-harboring bacteria was measured with quantitative real-time PCR(q PCR). The most prominent DGGE bands were excised and sequenced to study the phylotype composition of the diazotroph assemblage.In addition, the soil fungal community was studied in the same soil field study. The fungal community was analyzed using polymerase chain reaction(PCR) denaturing gradient gel electrophoresis(DGGE) targeting the 18 S r RNA gene of soil DNA and RNA. We further constrained the DNA- and RNA-derived fingerprints to the soil properties(i.e., total C, electrical conductivity, inorganic and organic N pools and p H) to determine which soil properties influenced the fungal community.The effect of organic management and fertilization in amino acid composition and concentrations was measured after application of the same amount of N as urea, alfalfa, rice straw or compost in organic and conventional soil. In an incubation study, for each treatment, three replicate jars were prepared and destructively harvested on days 1, 3, 7, 15, 28, 42 and 56 after the addition of N fertilizer, and water-extractable amino acid composition and concentrations, and inorganic and microbial N were measured.In order to establish the influence of fertilization on the bacterial and fungal communities, at the end of the incubation study the bacterial and fungal community structure was determined with PCR-DGGE targeting the 16 S and 18 S r DNA gene, respectively. Prominent DGGE DNA bands were sequenced to obtain taxonomic information on the members of the community. Microbial biomass, dehydrogenase activity and respiration were measured during the incubation to asses the effect on the soil quality and to track fertilizer decomposition dynamic.Nitrogenase activity was measured during the same soil incubation study to establish the potential N-fixation in organic soils and the effect of fertilizer addition. The acetylene reduction assay(ARA) is highly sensitive and was used in this study to measured nitrogenase activity. Other soil chemical and biological parameters were measured during the incubation to determine which soil parameters influence the most on the potential N-fixation. The diazotrophic community was analyzed at the end of the incubation using DNA-DGGE and targeting the nif H gene.The salient finds are as follows:(1) Ordination analysis of DGGE profiles revealed organic management affected the community structure in the greenhouse but not the open field; fertilization intensity may explain this divergent response, as indicated by the relevance of total C content to community structure. Quantitative PCR revealed that organic management increased the abundance and activity of diazotrophs. The soluble organic N concentration was higher in organically managed soils and during warmer months, and correlated with diazotroph abundance. Most identified sequences were from known diazotrophs, predominantly β-, γ- and α-proteobacteria. Twenty-four bands resembled Pseudomonas stutzeri and eight resembled Azoarcus sp. Our results show that the cultivation method controls the extent of the effects of season and organic management on diazotrophs, and that greenhouse cultivation can boost the effects of organic management on this community. Organic management intensified the positive effect of seasonal temperature on diazotroph abundance and activity, which may increase biological nitrogen fixation rates. In tandem, soil DNA and RNA analyses provide a comprehensive picture of the community.(2) The soil DNA fingerprints discriminated the fungal community by the management and cultivation types, while the c DNA fingerprints indicated a greater seasonal effect. Soil organic matter content, electrical conductivity and ammonium concentration could significantly explain most of the variation observed in the fingerprints. Sequencing of the DGGE bands revealed a greater presence of fungi affiliated to Pythium ultimum, Alternaria spp., Fusarium oxysporum, Sporisorium reilianum and Chaetomium globosum in conventional soils and Cordyceps gunnii in organic soils, with only partial overlapping of the sequenced 18 S r DNA and 18 S r RNA bands observed. The far-reaching influence of organic management is evidenced by the changes produced in both the total and theoretically active fungal populations. Changes in the fungal community due to greenhouse cultivation were associated with an increased salinity, producing a decrease in fungal biomass and increased bacterial activity. This suggests that the benefits derived from fungi in organically managed soils may be reduced by greenhouse cultivation practices. The c DNA-DGGE fingerprints could more sensitively detect changes in the patterns of the fungal community and were the only means to observe seasonal effects, demonstrating that joint analysis of soil DNA and RNA in the same study achieves a higher resolution than DNA-based studies alone, and allows the observation of otherwise unnoticed important changes in the fungal community.(3) The soil FAA pool was dominated by alanine, glutamic acid, glycine, isoleucine, leucine, phenylalanine, serine, tryptophan and valine. Organic and conventional soils did not significantly differ in their soil FAA composition and concentrations. However, the basicamino acids histidine and lysine were only present in organic soil, probably due a greater number of soil exchange sites in this soil. Urea significantly modified FAA composition, but only in organic soils, suggesting urea disrupts microbial structure and/or metabolic pathways in organic soils. Alfalfa and compost did not alter FAA composition and concentrations, indicating that any pulses of amino acids from these materials are short lived. On the contrary, straw significantly increased FAA concentrations after 15 days, coinciding with an increase in microbial biomass N. FAA concentrations remain low and have a largely constant composition in agricultural soils, even after long term organic management; however, some fertilizers can significantly alter FAA composition and concentrations, which may affect the importance of amino acid-N in the total N budget of plants. These findings warrant further research into the mechanisms controlling soil FAA composition and concentration in agricultural soils.(4) Microbial biomass and dehydrogenase activity were higher in the organic soil. The response of the microbial biomass and dehydrogenase activity to fertilizer addition was determined by the fertilizer type and was also dependent on management history. Urea addition decreased microbial biomass N in the organic soil and increased it in the conventional soil. In contrast, alfalfa addition increased microbial biomass N only in the conventional soil. Dehydrogenase activity was unaffected after urea addition and increased after alfalfa addition in both the organic and conventional soils. Straw addition increased significantly microbial biomass N and dehydrogenase activity but compost had little influence. Both bacterial and fungal communities differed significantly in the organic and conventional soils. Short term fertilization produced no changes in the microbial communities although addition of straw and alfalfa produced small variations in the fungal and bacterial fingerprints, respectively. Sequenced bands showed that bacterial and fungal species mainly affiliated to γ-Proteobacteria, Firmicutes, Acidobacteria and Ascomycota. Fertilization influenced soil chemical and biological properties; however, long term management is necessary to influence the microbial community. Our results showed that microbial properties response to short term fertilization are dependent on the management history and suggests that may be driven by the differences in their microbial communities.(5) The results showed a significant increase in potential N-fixation in organic compared to conventional soils. In the organic soils, alfalfa or compost addition increased the potential N-fixation in the organic soils but urea and straw decreased it in specific periods during the incubation. On the contrary, addition of alfalfa or urea in conventional soils did not produce significant changes. Dissolved organic carbon and p H strongly correlated with the potential N-fixation rate. The results from DGGE analysis revealed a simple community with no differences between organic and conventional soils and a limited impact from fertilizers, therefore, differences in the community structure can not explain the observe differences in potential N-fixation. The data suggest that rather than the community structure N-fixation was driven by the population size and activity. The study shows that in organic systems the potential for atmospheric N-fixation is greater than in conventional and addition of alfalfa or compost could increase it significantly; however, further research is needed to quantify actual N-fixation.This study addresses for first time the diazotrophic community in an organic vegetable production system, and the effect of cultivation method(greenhouse vs open field). In addition, the study measures for the first time the abundance of both present and active nif H-harboring bacteria in organic systems and the N-fixation potential. Other studies have previously evaluated the effect of land use on labile organic N pools but this study addresses for the first time the influence of an event fertilization on soil free amino acid concentration and composition.In summary, the N-related pools, microbial communities and processes varied greatly in organic and conventional soils but had a limited effect on soil FAA profiles. Addition of fertilizer influenced on the N pools and processes, but only had a minor influence on the microbial communities. The results indicate that while microbial communities requires long term management to be modified in the soil, the N-related pools and processes can be significantly altered by event fertilization. The study highlights that soil management can alter microbial ecology and processes involved in N transformations and N pools suggesting that management practices can influence the level of available N in soil and eventually may influence plant uptake.