Introduction
To meet increasing population demands, agricultural and forest ecosystems have been more intensively managed during the last five decades (Sengupta et al., 2015). However, the problem of soil degradation caused by long-term intensive management has become increasingly prominent (Guo, et al., 2010). Fertilization is the main practice employed in intensive management. For decades, many studies have reported that the increased application of single mineral fertilizers improved soil fertility and crop yields (Zeng et al., 2016; Wang et al.,2017b). However, this practice also accompanied various negative effects such as soil acidification, greenhouse gas emissions, nutrient losses, and deterioration of soil structure (Dalal, Wang, Robertson, & Parton. 2003; Zeng et al.,2016). Soil microbes responsible for most of the soil biochemical processes are also affected by changes in nutrient availability, pH and organic matter content resulting from heavy fertilization (Wang et al.,2017). It was reported that the input of mineral fertilizer reduced bacterial richness and disturbed soil microflora communities (Sun, Zhang, Guo, Wang, & Chu,2015). Reductions in microbial biomass was closely related to the duration and amount of N input in both field and lab-based studies (Treseder, 2008). Thus, long-term and large-scale application of mineral fertilizers is considered to be a key reason for reduced microbial biodiversity associated with intensive management (Wang et al.,2017). Therefore, land conservation and soil fertility recovery are very important to intensive agricultural system.
Bacteria-mediated fixation of C and N play an important role in sustaining soil fertility in agricultural ecosystems (Fan,2019; Kekulandara, Sirisena, Bandaranayake, Samarasinghe, & Suriyagoda, 2019). Autotrophic microorganisms along with algae contributed about 40% and between 4-10% of CO2 fixation in oceans and wetlands ecosystem, respectively (Cannon et al., 2001; Stanley, Johnson, & Ward, 2003). Many studies have focused on the assessing the importance of autotrophic bacteria in fixing atmospheric CO2 into soil OC(Ge et al., 2012;Wu et al., 2014;Yuan et al., 2015). Autotrophic bacteria in soil annually capture about 0.6–4.9 Gt C, which represents 0.5–4.1 % of total terrestrial C fixation (Falkowski et al., 2000). The C fixed is first imported into unstable OC pools as microbial biomass carbon (MBC) and dissolved organic carbon (DOC) (Ge et al., 2012). Biological N fixation (BNF) process has been considered, both economically and environmentally, as a source of N for plant growth. They play an important role on N supply for most ecosystems, especially in low-fertility soils (Norman & Friesen,2017). Globally, current estimates suggest that N fixed by BNF (~300Tg nitrogen yr-1) is much higher than that produced industrially (~125 TG nitrogen yr-1) (Kuypers , Marchant, & Kartal, 2018). It was estimated that soil N input via BNF accounted for 16% of global N2 input annually (Ollivier et al., 2011). Soil OC and total nitrogen (TN) contents were shown to increase significantly following inoculation with diazotrophic Azotobacter and Bacillus (Kheirfam, Sadeghi, Homaee, & Zarei, 2017). Introduction of one Azotobacter sp. strain, a free-living bacterium with excellent ability of N fixing, was estimated to save 50-75% of the mineral N and P fertilizer in 2 year field experiment (Dadrasan, Chaichi, Pourbabaee, Yazdani, & Keshavarz-Afshar, 2015). Besides enhancing N, C and P levels in agricultural systems, N-fixation bacteria could indirectly improve soil physical properties (Zhao, Qin, Weber, & Xu,2014), including declined soil density and increased water holding capacity, hydraulic conductivity and mean weight diameter(MWD)(Nisha, Kaushik, & Kaushik, 2007).
Bamboo is an important ecological, industrial and cultural resource. The total output value of the national bamboo industry reached 117.3 billion RMB in 2010 (National Bamboo Industry Development Plan 2011-2020).Moso bamboo (Phyllostachys pubescens ) covered 4.6778 million ha accounting for 73% of the total bamboo area in China by 2018 (the 9th national forest investigation) is a significant component of forest ecosystems (State Forestry Administration of the P.R. China, 2018). Due to its high economic return, Moso bamboo has received intensive management to enhance its productivity in the past few decades (Liu et al., 2011; Li et al., 2013). The intensive management (IM) practices employed with Mosobamboo forests are primarily annual fertilizer application and removal of understory herbs and shrubs. Farmers usually prefer to use mineral fertilizers which are more efficient and convenient than organic fertilizers, especially in mountain and hill land where farmer are difficult to practice. However, as with agricultural systems, long-time application of mineral fertilizers has resulted in the decline of soil fertility in bamboo plantation (Qin et al., 2017). As a result of observed ecological problems due to the sole use of inorganic fertilizers, combined applications of mineral fertilizers and manure (MCM) have been introduced into Moso bamboo management to prevent land from negative effects of mineral fertilizer.
The application of MCM has been proved to be a potentially superior land management practice than the application of mineral or organic fertilizers alone. Some field experiments confirmed that MCM played an important role in maintaining soil health, improving soil fertility, and promoting the restoration of biotic and abiotic soil properties (Wang, Lai, Wang, Pan, & Zeng, 2015). Meta-analysis and modelling data from upland soils and paddy-upland rotation soils across the major agricultural zones in China revealed that the MCM increased the SOC content and crop yields substantially (Jiang et al., 2018). The long-term fertilization experiment showed that application of MCM improved SOC significantly (Liang, Yang, He, & Zhou, 2011). It was demonstrated that SOC was positively correlated with crop yields following more than twenty years of continuous winter wheat–summer corn rotation cultivation (Yang, Zhao,  Huang, & Lv, 2015). There are several direct or indirect factors associated with MCM that contribute to improved crop yields. For example, the application of MCM has directly increased SOC and improved mineral N utilization efficiency by accelerating microbial SON mineralization activity (Pan et al., 2009). The higher SOC contents resulting from MCM treatment led to a greater cation exchange capacity (CEC) when compared with soils receiving with no or only inorganic fertilizers in a low-productivity paddy field (Mi et al., 2018). The MCM could potentially increase and modify microbial biomass, enzyme activities, or community composition by providing an OC energy source and nutrients in organic form (Zhao et al., 2016). MCM has generally had positive effects on bacterial CO2 and N2fixation. Fertilization increased cbbL abundance, with the highest cbbL copy number and RubisCO enzyme activity in NPK plus rice straw soil (Yuan et al., 2012). Long-term mineral NPK fertilization decreased the diversity of diazotrophic community, whereas NPK plus rice straw and NPK plus chicken manure treatments maintained the diversity of diazotrophic community (Liao, Li, & Yao, 2017). However, positive results have not always be observed. For example, Lin et al. (2018) reported that long-term application of inorganic fertilizer plus organic material (pig manure) suppressed the abundance and diversity diazotrophs and altered community structure, while inorganic fertilizer combined with plant residue (rice straw or radish) had no effect on the community structure of diazotrophs. The inconsistent results may be due to environmental heterogeneity and the type organic materials applied.
In the past few decades, long-term intensive management of Mosobamboo has been reported to cause soil deterioration, including soil erosion and nutrient leaching (Shinohara & Otsuki, 2015), soil acidification (Qin et al., 2017), and reduction of soil C and N storage (Li et al., 2013). It also caused a general decrease in microbial diversity and shifts of microbial community structure (Xu, Jiang, & Xu, 2008) and more specifically reduced the abundance and altered the community structure of arbuscular mycorrhizal fungi (AMF) (Qin et al., 2017). Soil CO2 and N2 fixation bacteria are considered sensitive to changes in soil nutrients, pH and organic matter content caused by heavy fertilization (Wu et al., 2014; Tang et al., 2017). It was observed that the abundance of CO2fixation bacteria in topsoil increased at the first 10 years of application of mineral fertilizer inMoso bamboo planation, and then decreased (Liu et al., 2018). However, the abundance and diversity of diazotrophic bacteria decreased at first and then increased (He et al.,2015). Thus, it is necessary to better understand how these bacterial groups respond to applications of MCM. We hypothesized that MCM could lead to a positive effect on CO2 and N2 fixation by microbial communities in Moso bamboo planation. The method of space-for-time substitution was used to establish a chronosequence of Moso bamboo stands with different durations of MCM management. We tracked changes in the genes cbbL andnifH , which respectively encode a component of ribulose-1,5-biphosphate carboxylase/oxygenase (RubisCO) and a nitrogenase reductase subunit, as these have been use previously to investigate the abundance and composition of CO2fixation (Videmšek et al., 2009; Yuan et al.,2015) and N2 fixation bacteria (Mmm, Marchant, & Kartal, 2018).