Organic substitutions aggravated microbial nitrogen limitation and decreased nitrogen-cycling gene abundances in a three-year greenhouse vegetable field
Graphical abstract
Introduction
Microorganisms are the dominant drivers of biogeochemical cycles. Their growth and activity are regulated by nutrient availability of carbon (C), nitrogen (N) and phosphorus (P) (Chen et al., 2018; Cui et al., 2018a; Deng et al., 2019; Wei et al., 2017) in addition to environmental factors. When available soil resources of C, N, and P meet microbial nutrient requirement, the maximum rate of microbial growth is achieved (i.e., stoichiometric balance) (Sterner and Elser, 2002). However, the nutrient requirement of microbial communities are rarely met by the considerable stoichiometric variations of soil resources, suggesting that microbial communities are limited generally by some certain elements (i.e. microbial nutrient limitation) (Cleveland and Liptzin, 2007; Mooshammer et al., 2014b). Microorganisms would change from growth to survival when available soil resources and microbial requirements do not match (i.e., stoichiometric imbalance) (Zechmeister-Boltenstern et al., 2015). Soil microorganisms respond to these stress conditions from stoichiometric imbalance by redistributing their resources to produce extracellular enzymes instead of promoting cell growth (Sinsabaugh et al., 2009). As a result, an increasing number of studies have been conducted to determine the status of microbial nutrient limitation by extracellular enzyme activity, including measuring β-1,4-glucosidase (BG) as an indicator of C energy demand, measuring β-1,4-N-acetylglucosaminidase (NAG) and leucine aminopeptidase (LAP) as indicators of N demand, and measuring alkaline phosphatase (AP) as an indicator of P demand (Moorhead et al., 2013; Rosinger et al., 2019; Sinsabaugh and Follstad Shah, 2012; Tapia-Torres et al., 2015).
A comprehensive meta-analysis of 690 independent experiments reported that organic amendment increased the contents of soil organic C and total N by 38.0% and 20.0% compared to mineral-only fertilization, respectively (Luo et al., 2018b). Organic fertilization could supply more balanced nutrient to microbial communities than chemical fertilization and altered microbial community structure (Cui et al., 2018a; Lin et al., 2019; Luo et al., 2018a). The composition of microbial community would determine its nutrient demands due to the fundamental differences in the element content and life strategy, e.g. bacteria with low C:N ratio (r-strategist) and fungi with high C:N ratio (K-strategist) (Fanin et al., 2013; Cui et al., 2018a; Deng et al., 2019). These alterations must affect the stoichiometric imbalances of C, N and P between microbial communities and their available soil resources. Recent studies have observed that organic fertilization shifted the microbial nutrient limitation (Zhang et al., 2019) or had negligible effects (Zheng et al., 2020a) on the basis of ecoenzymatic stoichiometry under maize–soybean rotation. Rosinger et al. (2019) demonstrated that bacterial growth, fungal growth and respiration were primarily limited by C in both grassland and forest soils.
The above analyzed challenges definitely illustrated that how microbial nutrient limitation responds to organic fertilization has not been fully elucidated, particularly for intensive vegetable ecosystems. According to the third national agricultural census in China, greenhouses covered an area of 1.3 million hectares at the end of 2016, which represents an increase of 241.0% over the end of 2006, ranking first among the total greenhouse vegetable production area in the world (NBS, 2017). The primary characteristics of intensive greenhouse vegetable production are high N fertilizer input (global average of 220 kg N ha−1 crop−1), high multiple cropping index, and frequent agricultural operations (Miao et al., 2011). At present, partially substituting chemical fertilizer with organic fertilizer has been widely employed to address food security, and to cope with soil acidification (Zhang et al., 2019), gaseous N losses, and N runoff and leaching problems (Miao et al., 2011; Zhou et al., 2019). It's urgent for researches on how these organic substitutions in intensive vegetable ecosystems would affect microbial nutrient limitation and then affect the microbial-mediated N cycle.
Some studies have linked different elements and determined that the stoichiometric ratios of substrate or microbial biomass can better explain the regulation of N-cycling processes than a single driver (Li et al., 2020; Zheng et al., 2020b). The size and flux of soil N pools and availability are clearly affected by the microbial-mediated N cycle (Dai et al., 2020). According to the consumer-driven nutrient recycling theory, these N-cycling processes are regulated by microbial biomass and activity, which is often limited by the resource availability of soil C, N and P (Sterner and Elser, 2002). For example, Cui et al. (2020b) found that N addition would aggravate microbial P limitation and then inhibit nitrification and denitrification gene abundances in semiarid agricultural ecosystems. The negative results of gene abundances were attributed to the inhibition of nitrifier and denitrifier activities due to P deficiency. Wei et al. (2017) found that P fertilization to P-limited soils enhanced gaseous N loss mainly through stimulating the growth of denitrifiers. Soil nitrification is expected to increase in relation to protein depolymerization with decreasing microbial N limitation (Wild et al., 2015). Furthermore, numerous findings have demonstrated that organic fertilization would increase gene abundances of nitrification and denitrification in favor of gaseous N loss, which was generally ascribed to improvements in microbial communities (Luo et al., 2018a; Ouyang et al., 2018a, 2018b; Sun et al., 2015). Given that microorganisms play an important role in the soil biogeochemical cycle, it is of great significance to further understand the relationship between soil microbial processes and resource constraints, for example, the nitrification and denitrification process, which are mainly performed by specialized microbial groups of nitrifiers and denitrifiers, respectively (Duan et al., 2019; Luo et al., 2018a).
Therefore, in this study, we assessed C, N and P stoichiometric imbalances and microbial nutrient limitations by measuring the C, N and P contents of both available soil resources and microbial biomasses, soil hydrolytic enzyme activities, and the biomarker gene abundances of nitrification (amoA-AOA and amoA-AOB) and denitrification (nirK, fungi nirK, nirS and nosZ) in a three-year greenhouse vegetable field at the Yangtze River Delta in China. We addressed three hypotheses: (i) The C, N and P stoichiometric imbalances between microbial communities and their available resources are still occurring due to intensified production of vegetable crops, (ii) organic substitutions would aggravate microbial nutrient limitation due to ecological stoichiometry of C, N and P, and (iii) organic substitutions would decrease the gene abundances of nitrification and denitrification, along with microbial N limitation in intensive vegetable agroecosystems.
Section snippets
Study site and experimental design
A three-year greenhouse vegetable field experiment was conducted from November 2016 to November 2019 in Nanjing, Jiangsu Province, China (32°01′N, 118°52′E), which belongs to the alluvial plain of the Yangtze River Delta. The climate is humid subtropical monsoon with an average air temperature of 15.4 °C and an annual rainfall of 1107 mm. Vegetables had been cultivated for nearly 12 years at this experimental site before 2016. The soil is classified as Haplic Luvisols with a texture consisting
Responses of stoichiometric imbalance and microbial homeostasis to fertilization strategies
The addition of chemical or organic N fertilizers significantly decreased the C:N imbalance than the control in 2018 and 2019 (P < 0.05). Organic substitutions caused no significant change in the C:N stoichiometric imbalance compared to chemical N fertilization in 2017 (P < 0.05; Fig. 1a). Different fertilization strategies caused non-significant variations of C:P imbalance from 2017 to 2019 (Fig. 1b). However, a relatively large rise in the number of N:P imbalances was observed in almost all
Stoichiometric imbalance, microbial nutrient limitation as affected by organic substitutions
Confirming to the hypothesis I, intensive vegetable filed showed obvious C, N and P stoichiometric imbalances, and the N:P imbalance was higher than the C:N (or P) imbalance among all treatments from 2017 to 2019 (Fig. 1, Fig. 5). To cope with these C, N and P imbalances, microbial communities adopted specific homeostatic regulations (Fig. 2), being strong homeostasis of N:P butweak homeostasis of C:N (or P) in the intensive vegetable field. Scott et al. (2012) reported that bacteria exhibited
Conclusions
Microbial communities presented a high N:P imbalance with strong N:P homeostasis, but a low C:N (or P) imbalance between microbial communities and their available resources with weak C:N (or P) homeostasis in greenhouse vegetable cropping. Organic substitutions aggravated microbial N limitation than chemical N fertilization due to the increase of C:N and N:P stoichiometric imbalances, which further decreased the gene abundances of nitrification and denitrification. However, the 1M1N treatment
Author’s statement
Haojie Shen: Investigation, Formal analysis, Writing – original draft. Qianqian Zhang: Data curation, Visualization, Revised version. Shuangge Zhu: Lab analysis. Pengpeng Duan: Data curation, Revised version. Xi Zhang: Project administration, Resources, Software. Zhen Wu: Data curation, Investigation, Resources. Zhengqin Xiong: Conceptualization, Funding acquisition, Supervision, Writing – review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We appreciate Editor and three anonymous reviewers for their valuable comments and critical evaluation on this manuscript. We thank Yanfeng Song for her assistance with experimental analysis. This work was supported by the National Natural Science Foundation of China (41977078). Authors declare no competing interests.
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