3 Results

3.1 Key soil attributes

The time since reclamation had a substantial effect on the studied soil key attributes (Fig. 2 ). Soil organic matter (SOM) significantly increased after 17 years since reclamation (Fig. 2A; χ2=0.68, p=0.410). Similarly, soil pH and EC increased significantly in the reclaimed plots with respect to the control plot (Figs. 2 B-C; χ2=22.432, p<0.001; χ2=19.557, p<0.001). Soil available potassium (AK) was significantly higher in the plots under reclamation than in the control plots (Fig. 2D; χ2=4.369, p=0.037). However, it was not clear whether the time since reclamation had an effect on the AK concentration in the soil. Both soil extractable phosphorus (OP) and total nitrogen (TN) were substantially lower in the reclaimed than in the control plots (Fig 2E-F; χ2=11.938, p=0.001; χ2=0.22, p=0.639). The enzymatic activity of soil urease (URA) in the soil was significantly higher in the plots under reclamation than in the control plots (Fig. 2G; χ2=16.518, p<0.001). Soil polyphenol oxidase (PPO) activity showed a clear increasing trend along the reclamation timeline (Fig. 2H; χ2=43.014, p<0.001). Yet, it was not clear whether the time since reclamation led to an increase in enzymatic activities of URA, dehydrogenase (DHG) and fluorescein diacetate hydrolase (FDA) in the soil (Fig. 2G and Figs 2I-J; χ2=1.16, p=0.281; χ2=0.065, p=0.799).

3.2 Taxonomic composition of the soil microbial communities (SMCs)

The most abundant phylum and genera identified in this study are shown in Figs. 3A and 3B, respectively. The phyla Thaumarchaeota ,Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Gemmatimonadetes, Patescibacteria , Planctomycetes ,Proteobacteria and Verrucomicrobia accounted for more than 95% of the total genetic sequences retrieved for each analyzed soil sample (Fig. 3). The relative abundance of the two most abundant phylum - i.e. Bacteroidetes and Proteobacteria decreased with increasing time after reclamation (Fig. 3A; χ2=18.339, p<0.001; χ2=26.530, p<0.001). The abundance of the third and fourth most abundant phylum i.e. Acidobacteria and Chloroflexiincreased following reclamation (Fig. 3A; χ2=15.437, p<0.001; χ2=42.547, p<0.001). Moreover, the abundance of the phylum Thaumarchaeota ,Actinobacteria and Planctomycetes also increased with the time after reclamation (Fig. 3A; χ2=0.000, p=0.993; χ2=9.862, p=0.002; χ2=1.346, p=0.246), but the abundance of the phylum Verrucomicrobia, Cyanobacteria , Dependentiae , Patescibacteria , FBPand Firmicutes decreased. The relative abundance of other identified phylum is shown in supplementary material – Fig. S2.
Regarding the most abundant SMCs’ genera found (Fig. 3B), the abundance of the genera RB41 and UTCFX1 increased significantly with the time after reclamation (Fig. 3B; χ2=18.110, p<0.001; χ2=43.022, p<0.001). Nonetheless, other genera, such as Flavobacterium ,Pedobacter , Sphingomonas and Luteimonas (Fig. 3B; χ2=21.616, p<0.001; χ2=13.077, p<0.001; χ2=25.592, p<0.001; χ2=12.872, p<0.001) showed the opposite trend. The abundance of the less abundant genus is shown in supplementary material – Fig. S2.

3.3 Molecular ecological networks (MENs)