bulk soil and rhizosphere
The different clades from phylum to genus level in both the bulk soil and rhizosphere were visualized by LEfSe (Figure 4). Specifically, in the bulk soil (Figure 4a, Table 1), the LDA score (LDA score > 4.0,p < 0.05) identified phyla Firmicutes and Bacteroidetes, as well as class Actinobacteria (order Bifidobacteriales, family Bifidobacteriaceae and genus Bifidobacterium ) in C_NS, as well as class Alphaproteobacteria (genusSphingobium ) in F_NS as differentially abundant bacterial taxa. Moreover, as the same classes were found in the bulk soil: Actinobacteria (family Microbacteriaceae and genusMicrobacterium ) in C_NS and Alphaproteobacteria (order Sphingomonadales, families Sphingomonadaceae and Xanthobacteraceae) in F_NS were found to be significantly enrichment in the rhizosphere. Moreover (Figure 4b, Table 2), the LDA score (LDA score > 3.0, p < 0.05) indicated significant enrichment of C_NS for class Eurotiomycetes. The phylum Rozellomycota and class Sordariomycetes in F_NS were among the most differentially abundant fungal taxa in bulk soil samples. OTUs representing the fungal class Leotiomycetes were significantly enriched in C_NS while Tremellomycetes and Dothideomycetes were detected in F_NS rhizosphere.
In addition, specific microbial clades were also detected in the reassembled soil bacterial communities (Figure 4c, Table S3). Briefly, the LDA score (LDA score > 4.0, p < 0.05) indicated that class Gammaproteobacteria (family Rhodanobacteraceae) and Bacteroidia dominated F_S, thus forming bulk-soil bacterial communities. In the rhizosphere, C_S was selectively enriched with class Oxyphotobacteria, Gammaproteobacteria (genus Lysobacter ) and phylum Patescibacteria, while F_S showed great abundance of phylum Actinobacteria. Importantly, the LDA score (LDA score > 3.0, p < 0.05) suggested that no fungal clade was significantly enriched among the reassembled fungal communities.
3.4 Influences of continuous cropping and natural fallow practice on rhizosphere effect
This study documented the core microbiomes in rhizosphere of N. tabacum (Table S4). From the total of 1 790 clustered bacterial OTUs found in rhizosphere, 432 were consistently present among all samples. Those 432 OTUs, classified up to genus level (Figure S4) were mainly involved in Sphingomonas (7.71%), Luteimonas (6.16%), Rhodanobacter (5.43%), and Arthrobacter (4.67%). Moreover, the core of the fungal microbiome was composed of 78 OTUs, which represented 13.2% of the total OTUs. Fusarium (15.52%),Penicillium (9.2%), and Mortierella (6.92%) were the most abundant genera (Figure S4).
In addition to the core microbiomes ofN. tabacum found in rhizosphere, the specific differences of the rhizosphere effect were investigated. For N. tabacumplants grown in CCS, 519 (18.1%) and 425 (19%) bacterial OTUs were found only in the rhizosphere of C_NS and C_S, respectively (Table S5). Compared with the bulk soil, the LDA score (LDA score > 3.0,p < 0.05) indicated that the orders Chloroplast and Corynebacteriales, genusSphingopyxis in C_NS, as well as class Oxyphotobacteria, order Micrococcales and genus Sphingopyxis in C_S were found only in the rhizosphere. Moreover, 183 (33.6%) fungal OTUs related to the order Saccharomycetales and family Cladosporiaceae were found only in rhizosphere samples compared with those in the bulk soil in C_NS (LDA score > 2.0,p < 0.05). Moreover, 194 (58.1%) fungal OTUs related to class Dothideomycetes and genusSporobolomyces were only detected in the rhizosphere of C_S (LDA score > 2.0, p < 0.05). For N. tabacum plants grown in FS, 403 (19.8%) and 279 (23.1%) bacterial OTUs were only found in rhizosphere in F_NS and F_S, respectively (Table S6). Compared with the bulk soil, the LDA score (LDA score > 3.0, p < 0.05) indicated that phyla Cyanobacteria and Firmicutes, order Corynebacteriales, and genus Nocardioidesin F_NS as well as phyla Cyanobacteria and Actinobacteria, family Lachnospiraceae, and genus Bacillus in C_S were only found in rhizosphere samples. In addition, 192 (28.5%) fungal OTUs related to orders Venturiales and Capnodiales, and family Sympoventuriaceae were only found in the rhizosphere when compared with those in the bulk soil in F_NS (LDA score > 3.0, p < 0.05). Although 83 (30.6%) fungal OTUs were found only in the rhizosphere of F_S, no species was indicated under such filter criteria (LDA score > 2.0, p < 0.05).