Overall and alpha diversity analysis of sequencing data
Bacterial community structure diversity of rhizosphere soils from four peanut cultivars with or without salt stress was carried out by 16S rRNA gene sequencing. Salt-resistant (big-grain peanut Huayu25, small-grain peanut Luhua12) and susceptible (big-grain peanut Huayu33, small-grain peanut Huayu20) peanut cultivars were used in this study. Bulk soil group without plants was collected and designated as control bulk soil (CB). We combined the controlled rhizosphere soil groups from Huayu25, Luhua12, Huayu33, and Huayu20 to controlled rhizosphere soil of resistant big-grain peanut (CRBR), resistant small-grain peanut (CRSR), susceptible big-grain peanut (CSBR), and susceptible small-grain peanut (CSSR), respectively. And salt-treated rhizosphere soil groups from Huayu25, Luhua12, Huayu33, and Huayu20 were designated as salt-treated rhizosphere of resistant big-grain peanut (SRBR), resistant small-grain peanut (SRSR), susceptible big-grain peanut (SSBR), and susceptible small-grain peanut (SSSR), respectively. After filtering using the QIIME software (Quantitative Insights into Microbial Ecology, v1.8.0), 1,357,465 sequences passed quality screening and most of which were between 401 and 420 bp (Fig. 1A and Table S1). Operational taxonomic units (OTUs) were generated with a 97% threshold identity, and a total of 6,429 OTUs were detected in peanut rhizosphere soils (Table S1).
To analyze community richness and diversity, alpha diversity analysis was performed. Alpha diversity indices (Sobs, Shannon and Simpson, Chao1 and ace, and coverage) which can reflect the community richness and diversity of the bacterial community were also performed (Fig. 1B and Table S2). The Good’s coverage index is 0.997 ± 0.001, indicating an adequate sequencing depth, which is further proved by other alpha diversity analysis. Rarefaction curve analysis showed that in the process of population sampling, the rate of increase of new species increased with the increase of sample size and then gradually leveled off, indicating that the sequencing depth of this peanut rhizosphere soil was high enough to analyze the community richness (Fig. 1C). Rank abundance curve showed that all the rhizosphere soil samples had high species evenness and homogeneity (Fig. 1D). All the results showed that alpha diversity revealed no significant difference among the different soil samples of the peanut rhizosphere.
Peanut Rhizosphere Bacterial Community Structure Taxonomic Analysis
To further analyze the bacterial community structure, we analyzed the abundance distributions of each soil group at five levels (phylum, classes, orders, families, and genus). The bacterial community of peanut is mainly composed of Actinobacteria, Proteobacteria, Chloroflexi, Acidobacteria, and Cyanobacteria at the phylum level, accounted for more than 80% of total bacterial sequences (Fig. 2A). Further structure analysis showed that bacterial community abundance indeed altered with salt stress, whereas no significant differences were detected among the four peanut cultivars. Cyanobacteria and Proteobacteria increased in most salt-treated soils, while Acidobacteria decreased in all the salt-treated soils compared to the controlled rhizosphere soils. Among them, the abundance of Cyanobacteria in SRBR slightly higher than that of controlled rhizosphere soils, while lower than that of other salt-treated rhizosphere soils (Fig. 2A and Fig. S1). Furthermore, Chloroflexi were the dominant bacteria in the bulk soils, which decreased in peanut rhizosphere soils (Fig. 2A and Fig. S1). At the class level, most of the bacterial community belonged to Actinobacteria, Alphaproteobacteria, Acidobacteria, Cyanobacteria, and Betaproteobacteria (Fig. 2B). The presence of Acidobacteria and Betaproteobacteria decreased in salt-treated soils, whereas numbers of Alphaproteobacteria and Cyanobacteria increased in these soils. Gammaphaproteobacteria increased in all peanut rhizosphere soils than in the bulk soils (Fig. 2B). Beneficial bacteria Rhizobiales were the most abundant order in peanut rhizosphere soils and Micrococcaceae predominated in peanut rhizosphere soils at the family level (Fig. 2C, D).
A thorough investigation at the genus level was performed in our study and the top 10 most abundant genera were shown by Wilcoxon rank-sum test in Fig. 3A. Pseudarthrobacter, norank_c__Acidobacteria, Sphingomonas, and norank_c__Cyanobacteria were predominantly found in the peanut rhizosphere soils. Many sequences named as “norank” or “unclassified” were unidentified species but were abundant in peanut rhizosphere soils, demonstrating that the peanut soils remained a challenging reservoir of biodiversity which needed to further study (Fig. 3A and Excel S1). Salt treatment increased the quantities of Rhizobium but reduced the numbers of RB41, Gaiella, norank_o_Gaiellales, and norank_c__Acidobacteria (Fig. 3A). Pie chart analysis was also performed to provide the comprehensible depiction of bacterial groups present in four peanut cultivars with or without salt stress. The top 10 most abundant genera were identical in different rhizosphere soils, whereas the proportions and abundance were significantly different (Fig. 3B and Fig. S2). Thus, salt stress can rapidly cause the shift of bacterial abundance distributions and the enrichment of some specific bacterial species in peanut rhizosphere soils.
Clustering Analysis Of Bacterial Community
Beta diversity analysis and clustering analysis were performed to check the similarities and dissimilarities among different soil groups. Principal component analysis (PCA) and principal co-ordinates analysis (PCoA) analysis showed that the bacterial community compositions of different soil groups were diverse (Fig. 4A, B). And bacterial community compositions of four salt-treated soil groups were more similar to each other but farther apart from the four controlled rhizosphere soil groups in different coordinates (Fig. 4B). Cluster analysis in the form of a hierarchical tree showed that the bacterial community structures of salt-treated and controlled rhizosphere soils were diverse, whereas the duplicate samples in the same soil group were similar to each other as other beta diversity analyses (Fig. 4C).
Pseudarthrobacte were the most abundant genera in the peanut rhizosphere and 35% of the top 20 richest genera belonged to the phylum Actinobacteria in the phylogenetic tree (Fig. 5A and Fig. S3). Heat map analysis showed that Flavobacterium, Devosia, and Rhizobium at the genus level occurred relatively higher abundance in salt-treated rhizosphere soils (Fig. 5B). Thus, all the results show that salt stress leads to the change of bacterial community and enrichment of Flavobacterium, Flavobacterium, and Rhizobium at the genus level, which may be beneficial to peanut survival and salt resistance as other PGPRs.
Specific phylotypes of rhizosphere bacterial community modulated by salt stress
Linear discriminant analysis (LDA) effect size (LEfSe) was employed to identify specific phylotypes of peanut rhizosphere bacterial community responding to salt stress from phylum to genus level in cladograms (Fig. 6 and Fig. S4). LEfSe analysis confirmed the significant enrichment of Rhizobiales (from order to genus) and Flavobacteriia (from class to genus) in the salt-treated group (SSBR) and enrichment of Chloroflexi (from phylum to order) in the bulk soil (CB) found by community structure taxonomic analysis (Figs. 2, 3, and 6). In the bulk soil (CB), additionally found the enrichment of Firmicutes (from phylum to genus, namely p__Firmicutes, c__Bacilli, o__Bacillales, f__Bacillaceae, and g__Bacillus) and TK10 (from class to genus, c__TK10, o__norank_c__TK10, f__norank_c__TK10, and g__norank_c__TK10). In peanut rhizosphere soils of different treatments, the predominant bacterial community was diverse. In controlled rhizosphere soils, phylum Acidobacteria dominated in CRBR, and phylum Gemmatimonadetes and Saccharibacteria dominated in CSSR, while class Acidimicrobiales and Clostridiales specifically elevated in CRSR and CSBR, respectively (Fig. 6). After salt stress treatment, Rhodobacterales and Xanthomonadales dominated in SRBR, and phylum Cyanobacteria and genus Sphingomonas were predominant in SRSR (Fig. 6). Additionally, in rhizosphere soils of salt susceptible peanut cultivars, SubsectionIII (from order to genus, namely o__SubsectionIII, f__FamilyI_o__SubsectionIII, and g__unclassified_f__FamilyI_o__SubsectionIII) and family Intrasporangiaceae were relatively abundant in SSSR. The bacterial community structure shows significant specificity in different treatments, which may be associated with their survival abilities or potential roles under salt stress.
Metabolic Functional Features Prediction Of Peanut Rhizosphere Bacterial Community
We then predict the function of bacterial population metabolism based on the known microbial genomic data. The cluster of orthologous groups (COG) database analysis showed that four metabolic functions (namely amino acid transport and metabolism, lipid transport and metabolism, energy production and conversion, and posttranslational modification, protein turnover, chaperones) were more abundant in salt-treated soil groups (Figs. 7A, B and Fig. S5). Furthermore, salt stress-related classifications including signal transduction mechanism and inorganic ion transport and metabolism were also enriched in these salt-treated soils (Fig. 7A). These vigorous metabolic functions may be associated with salt stress tolerance of bacterial communities in the salt-treated soils.
Qpcr Of Specific Bacterial Groups
To further verify the 16S rRNA gene sequencing data, qPCR assay further confirmed the changes in the abundance of the major bacterial phyla (Actinobacteria, Proteobacteria, Chloroflexi, Acidobacteria, and Cyanobacteria) and salt-induced bacteria Rhizobium in all soil samples. The numbers of phylum Cyanobacteria and Alphaproteobacteria and beneficial bacteria Rhizobium were significantly higher in salt-treated peanut rhizosphere soils, whereas Acidobacteria was relatively lower in these soil samples as the taxonomic analysis in Fig. 2A (Fig. 8). And Chloroflexi were enriched in the bulk soils, and gradually decreased in peanut rhizosphere soils (Fig. 2A and 8D). Most of the results were consistent with the 16S rRNA gene sequencing analysis. Taken together, all the results indicate that Cyanobacteria and Alphaproteobacteria as predominant phyla in salt-treated soils.