3.5 The cbbL- and nifH-containing bacterial diversity and their relationship with soil properties
Soil microbial diversity was employed as a third indicator of microbial response to IM. The diversity of CO2 and N2 fixation bacteria responded similarly to IM (Table 2). It was observed that IM10 was unique in both topsoils and subsoils with respect to diversity indices of CO2 fixation bacteria, with both Shannon and Evenness values being lower level compared with the remaining treatments (P < 0.05), while the Simpson index displayed an opposite trend. Similar trends were observed for diversity indices of diazotrophic bacteria in topsoils, while no differences were observed in subsoils. Two-way ANOVA analysis of variance revealed that the diversity of cbbL - andnifH -containing bacteria was significantly affected by IM time and even more so by soil depth (Table 2). Correlation analysis between diversity index and soil properties based on same layer revealed that evenness and Shannon indices of cbbL -containing bacterial were positively correlated only with AK (P = 0.016) in the topsoils and with AP (P = 0.015) in the subsoils (Table 3), respectively. However, more soil factors were positively correlated with diversity index of nifH -containing bacterial, including evenness index (E) with SOC (P = 0.021), TN (P = 0.033) and AN (P = 0.040) in topsoils, and with SOC (P = 0.020), TN (P = 0.014) and AN (P = 0.023) in subsoils. Soil TN and AN were also positively correlated with Shannon index in subsoils.
  1. Discussion
  2. Alteration of soil properties during 20 year of IM
Soil pH usually decreases as a result of long term fertilization with high rates of mineral fertilizer (Schroder et al.,2011). It is encouraging that soil pH was stable in response to MCM management in this study. Soil nutrients usually accumulate with increasing duration of IM because of annual fertilizer applications (Tan et al.,2013). This study demonstrated that application of MCM resulted in enhanced soil fertility with the exception of NH4+-N. The increased NO3--N and decreased NH4+-N over time might be the result of enhanced nitrification, a commonly observed response to intensive agriculture (Bi et al., 2017). This improved fertility is mainly attributed to direct input of mineral fertilizers and indirect supplement of organic fertilizers decomposition. Soil OC varied over time, increasing sharply at IM6, returning to a lower level similar to CK for more than 10 years, and finally recovering somewhat at IM20. It has been shown that organic fertilizer usually improves SOC in long-term studies (Han et al.,2018). We also detected an increase of SOC after 20 years of IM, although levels did fluctuate during the preceding 15 years. The dynamics of SOC depends on the balance of OC input and its mineralization. The sharp increase at the first stage was reflected the large amount of manure applied that did not decomposed rapidly. However, mineralization accelerated with the improvement of soil microbial community during the second stage, so that SOC then dropped to the same level as CK. Soil δ13C is closely related to SOC dynamics and is an important index to study the history of reconstructed plant communities, determine the SOC source, soil quality, and soil C sequestration rates (Mendez-Millan et al., 2014; Zhang et al.,2015). Soil δ13C increased with duration of IM and reached the maximum at IM15 (Fig. 1). This increase in δ13C may be attributed two factors. One was that the lighter isotopes of 12C were easier to volatilize via organic matter decomposition compared to those containing13C, which resulted in the increased relative abundance of 13C (Guillaume, Muhammad, & Kuzyakov, 2015). It is possible that the chemical bonds containing 12C are easier to break down than 13C in enzymatic reaction (Powers & Schlesinger, 2002). Another one was that input pig manure was rich in13C because pig food contain C4 plant of maize having higher δ13C (ranged from-17‰ to -9‰) than C3 plant (ranged from-32‰ to -22‰) (Bai et al., 2012; Farquhar, Ehleringer, & Hubick, 1989).