In the above section, we investigated bacterial diversity in frog intestines; however, soil microbiota, which also share a symbiotic relationship with frogs, are also worth exploring. First, we found that the number of ASVs for soil bacteria in frog habitats was twice that of gut bacteria, suggesting that the diversity and richness of soil bacteria is significantly greater than that of gut bacteria. Among different habitats, bacterial community richness was significantly higher in NS than in AS and SS, but soil bacterial diversity did not differ significantly. Xiaomei Yi et al. conducted the first comprehensive study on the structure and function of soil microbial communities in rice field (RF) and showed that RF significantly increased the diversity and richness of bacterial and fungal communities [27]. This may be due to the increase in frog feces with increasing cultivation time, which favors the growth of various microorganisms. Studies on soybean soil have also reported that bacterial species richness is significantly higher in agricultural soil than in native soil, but diversity does not differ significantly between the two soil types [25]. In our study, we reached a conclusion contrary to the two aforementioned studies, possibly because the intense activity of frogs in HS and SS suppressed soil bacterial richness.
Further research indicated that the type of frog habitat had a significant effect on soil bacterial β-diversity. Interestingly, a study by Pérez-Jaramillo et al. showed that there were significant differences between soybean agricultural soils and native soils [25]. We found that the dominant phyla of bacteria in frog habitat soil were mainly Proteobacteria, Bacteroidetes, Acidobacteria, and Chloroflexi, among which Firmicutes were most abundant in NS; Ignavibacteriae were much higher in AS than in other soils. However, their differences at the phylum level were not significant. The research of Xiaomei Yi et al. found that Proteobacteria, Acidobacteria, and Chloroflexi were the dominant bacterial communities in rice-frog cultivation (RF) soil, and the specific bacterial taxa enriched in RF played an indispensable role in organic matter decomposition and soil C, N, and P transformation processes [27]. They were also identified in our samples. In addition, research has shown that Acidobacteria have a higher relative abundance in native soil than in soybean agricultural soil [25], and in our study, Acidobacteria were found to be highest in SS (63.28%). Acidobacteria are generally considered to be oligotrophic, and the abundance of acidic bacterial species in soil [63], as well as their diversity in metabolic characteristics, make them a potentially important group in soil nutrient cycling [64]. This may be due to the fact that frog culture promotes the enrichment of Acidobacteria in soybean soil.
At the genus level, we observed significant differences in the dominant genera Bacillus, Nitrosospira, and Geobacter. Another study found that the core bacterial genera in agricultural soybean soil include Rhizobium, Bradyrhizobium, Mesorhizobium, and Sphingomonas, with a considerable portion comprised of nitrogen-fixing bacteria [25]. In our study, Sphingomonas and Bradyrhizobium were significantly higher in abundance in soybean soil compared to other soil types, suggesting that soybean soil in frog farming is rich in nitrogen-fixing bacteria.
Furthermore, we constructed bacterial ecological networks in frog habitat soils. This research revealed that the HS bacterial network structure was the most complex, with the tightest connections between bacteria. As the soil with the most active frog activity, this may be due to frog excreta and food providing a nutrient source for soil microbes. Ignavibacterium and Sphingomonas were detected as the most critical taxa (strongest interaction) in HS, with the stability and construction of the bacterial network structure primarily relying on them. In terms of bacterial community modularity, SS exhibited the most distinct pattern, while also possessing the highest number of key taxa, including Geobacter, Sphingomonasa, Sphingobacterium, and Bradyrhizobium. These key taxa displayed a significantly positive correlation with other genera. Previous research found that the interactions between bacterial taxa in native soil environments were more complex than those in soybean agricultural soil, but soybean soil favors the establishment of nutrient-rich organisms [25]. Based on these findings, we hypothesize that bacterial network modularity is shaped by soybean soil in frog farming, which in turn allows functionally relevant bacterial species to more easily establish themselves in soybean agricultural soil, such as Geobacter, Sphingomonas, Sphingobacterium, and Bradyrhizobium.
Bacteria play a crucial role in soil nutrient cycling, and their functions determine soil fertility and microbial vitality to some extent [65]. According to FAPROTAX, we predicted that 35.6% of ASVs possess potential ecological functions. Most functions in soybean soil (SS) were significantly higher than in the other two soil types, such as nitrogen_respiration, iron_respiration, sulfur_respiration, and nitrogen_fixation. Iron_respiration was notably higher in SS than in other soils, which might be closely related to the presence of abundant nitrogen-fixing bacteria in that environment. Furthermore, we found that many functions were significantly higher in HS than in the other two soil types, including hydrocarbon_degradation, dark_hydrogen_oxidation, methanotrophy, methylotrophy, and dark_sulfide_oxidation. HS provides favorable conditions for bacteria associated with dark_hydrogen_oxidation and dark_sulfide_oxidation due to its moist and light-avoiding environment. In contrast, NS areas lack interference from plants and animals such as frogs, resulting in generally lower functional microorganisms and weaker soil microbial metabolic activity compared to other soils. Regarding the assembly and ecological niche width of soil bacterial communities, we applied the neutral model to fit bacterial communities in different frog habitats. We found that the dispersal limitation of NS bacterial communities was much more severe. NS is considered to be heterogeneous and discontinuous soil for microbes [66]. In contrast, due to the influence of soybean plants (SS) or frog habitation (AS), the soil environment is more uniform. As a result, more ecological niches exist in NS soil. Additionally, deterministic mechanisms had a more significant impact on the community composition of SS, likely because deterministic processes have a more substantial influence on soybean soils with a narrower niche width.