Influence of Salt Stress on Growth of Spermosphere Bacterial Communities in Different Peanut (Arachis hypogaea L.) Cultivars

Abstract

Background: Exposure of seeds to high salinity can cause reduced germination and poor seedling establishment. Improving the salt tolerance of peanut (Arachis hypogaea L.) seeds during germination is an important breeding goal of the peanut industry. Bacterial communities in the spermosphere soils may be of special importance to seed germination under salt stress, whereas extant results in oilseed crop peanut are scarce.

Methods: Here, bacterial communities colonizing peanut seeds with salt stress were characterized using 16S rRNA gene sequencing.

Results: Peanut spermosphere was composed of four dominant genera: Bacillus, Massilia, Pseudarthrobacter, and Sphingomonas. Comparisons of bacterial community structure revealed that the beneficial bacteria (Bacillus), which can produce specific phosphatases to sequentially mineralize organic phosphorus into inorganic phosphorus, occurred in relatively higher abundance in salt-treated spermosphere soils. Further soil enzyme activity assays showed that phosphatase activity increased in salt-treated spermosphere soils, which may be associated with the shift of Bacillus.

Conclusion: This study will form the foundation for future improvement of salt tolerance of peanuts at the seed germination stage via modification of the soil microbes.

Keywords: bacterial community diversity; peanut (Arachis hypogaea L.); peanut cultivars; salt stress; spermosphere.

Figure 1

Overall sequence data and alpha diversity analysis. (A) Operational taxonomic units (OTUs) of different soil groups. CS, control bulk soil; SS, salt-treated bulk soil; CS25, controlled spermosphere soil around Huayu25 (salt-resistant peanut cultivar); CS20, controlled spermosphere soil around Huayu20 (salt-susceptible peanut cultivar); SS25, salt-treated spermosphere soil around Huayu25; SS20, salt-treated spermosphere soil around Huayu20. (B) Rarefaction curve analysis showing the gene sequencing depth. (C) Species accumulation curves showing the rate of increase of new species with the increase in sample size. “+” represents extreme outliers. Single red box reflects the total number of species in the sample, and all the red boxes form the accumulation curve. The single green box reflects the number of common species in the sample, and all the green boxes form the common quantity curve. (D) Rank abundance curve showing the relative species abundance and evenness. The length of the polyline on the horizontal axis reflects the OTU numbers and represents the richness of the bacterial community. The flatness of the polyline reflects the evenness of the bacterial community composition.

Figure 2

Bacterial community structure in the peanut spermosphere soils and the bulk soils at the phylum, class, order, and family level. (A) Percent of taxa at the phylum level in the peanut spermosphere soils and the bulk soils. The relative abundance was calculated by averaging the abundances of three duplicates in each soil group. (B) Percent of taxa at the class level in the peanut spermosphere soils and the bulk soils. The relative abundance was calculated by averaging the abundances of three duplicates in each soil group. (C) Percent of taxa at the order level in the peanut spermosphere soils and the bulk soils. The relative abundance was calculated by averaging the abundances of three duplicates in each soil group. (D) Percent of taxa at the family level in the peanut spermosphere soils and the bulk soils. The relative abundance was calculated by averaging the abundances of three duplicates in each soil group.

Figure 3

Bacterial community diversity analysis through bar charts and heatmap. (A) Percent of taxa at the genus level in peanut spermosphere soils and the bulk soils is visualized using bar charts. Samples are clustered according to the similarity among their constituents and arranged in vertical order. The shorter the branch length between samples, the higher the similarity of the two samples. (B) The relative abundance of the top 20 abundant genera is visualized using a heatmap. Samples are clustered according to the similarity among their constituents and arranged in a horizontal order. The taxa are also clustered according to the degree of similarity distributed among different soil samples and arranged in a vertical order. The red rectangle represents the more abundant genera and the blue rectangle represents the less abundant genera.

Figure 4

Beta diversity analysis. (A) Principal component analysis (PCA). Two principal components (PC1 and PC2) of PCA are shown in the coordinates. The same color points belonged to the same soil group are closer to each other and the samples from different soil groups are farther apart. (B) Unweighted pair-group method with arithmetic mean (UPGMA) analysis is clustered according to samples’ similarity. The longer the branch length between samples, the more variable the two samples are. (C) Analysis of similarities (ANOSIM) revealed the variation in the composition (Bray–Jaccard distance) of spermosphere or bulk soil bacterial communities.

Figure 5

Cladogram showing specific phylotypes of bacterial community compositions of peanut spermosphere responding to salt stress. Circles indicate phylogenetic levels from phylum to genus (from the outer circle to the inner circle). The diameter of each circle is proportional to the abundance of the bacterial group.

Figure 6

Metabolic functional features of the peanut spermosphere bacterial community. (A) Bar chart showing the relative abundance and diversity of functional groups in various peanut spermosphere soil groups and bulk soil groups in the context of the Cluster of Orthologous Groups (COG) database. Different COG groups are displayed in different colors, as listed in the right. (B) The Kyoto Encyclopedia of Genes and Genomes (KEGG) database showing the relative abundance and diversity of functional groups in various peanut spermosphere soil groups and bulk soil groups.

Figure 7

Soil enzyme activities of the peanut spermosphere soils and the bulk soils. Soil (A) invertase, (B) calatase, (C) neutral phosphatase, and (D) urease activities of the peanut spermosphere soils and the bulk soils. Error bars indicate the SEM (n = 3). One-way ANOVA Duncan’s test. Different lowercase letters represent different significance.

Figure 8

Quantification of predominant phyla or genera in the peanut spermosphere soils and the bulk soils. Quantification of (A) Alphaproteobacteria, (B) Betaproteobacteria, (C) Firmicutes, (D) Actinobacteria, (E) Bacteroidetes, and (F) Bacillus in various peanut spermosphere soils and bulk soils. Error bars indicate the SEM (n = 3). One-way ANOVA Duncan’s test. Different lowercase letters represent different significance.