Comparative analysis of actinorhizal nodule and associated soil microorganism diversity and structure in three Alnus species

Abstract

Background: Due to the importance of biological nitrogen fixation in terrestrial ecosystems, actinorhizal symbiosis has attracted more and more attention. Alders (Alnus) are important actinorhizal plants, but little is known about the diversity of symbiotic microbiota in the actinorhizal nodules. In addition, it remains unclear about the influence of the host species and habitats on the microbial community of alder root nodules and rhizospheric soils.

Methods: In this study we sequenced the hyper-variable regions of the 16S rRNA from the root nodules and their rhizosphere soils of three alder species (Alnus mandshurica, A. sibirica, A. japonica) in northeastern China to explore the diversity, composition, network association, and nitrogen cycling pathway of the microbial communities in the actinorhizal nodules and associated soils.

Results: The results showed that the microbial community α-diversity decreased significantly from the associated soil to the root nodule, and the microbial diversity in the root nodule of A. sibirica was not affected by the habitats. The dominant microbe genus in alder nodules was Frankia, whose abundance was significantly higher than that in associated soil samples. Furthermore, the abundance of Frankia was affected by alder tree species, but not by the habitats. The most significant taxon in the nodules of all the three alders was Frankia genus, which was negatively correlated with other six genera of microbes. The main function of microorganisms in alder nodules is nitrogen fixation, which is not affected by tree species and their habitats.

Conclusion: These findings suggest that the host determines the microbial community composition in the root nodule of three alders. This study provides valuable insights into the effects of alder species and habitats on the microbial communities of alder nodules and associated soils.

Keywords: 16S rRNA; Alnus; Frankia; root nodules; symbiosis.

Figure 1

Sampling sites of Alnus species. The red zone includes Jilin and Liaoning provinces in northeast China. Green dot: A. mandshurica, Yellow dot: A. japonica, Pink dot: A. sibirica, and marked with place names in the map.

Figure 2

Microbial community α-diversity in root nodules and associated soils of different alders. (A) Comparison of microbial community Chao index between root nodules of three different alder species and associated soils. (B) Comparison of microbial community Shannon index between root nodules of three different alder species and associated soils. The Student’s t-test was used to compare the two groups. Am_nodule, As_nodule, and Aj_nodule represent the root nodules of A mandshurica, A sibirica, A japonica, and Am_soil, As_soil, and Aj_soil represent the associated soils of A mandshurica, A sibirica, A japonica, respectively. The same applies below. Asterisks indicate significant differences between samples, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Microbial community β-diversity and constituent structure in root nodules and associated soils of three alder species. (A) PCoA analysis of six different groups on OTU level. (B) Relative abundance of the top ten dominant phyla in microbial community of six different groups. (C) Difference analysis of the abundance of ten dominant phyla between nodule and soil. (D) Relative abundance of the top ten dominant genera in Actinobacteria phylum of microbial community. PERMANOVA was used to analyze the impact of different grouping factors on sample differences. Only differentially abundant phyla were shown in Figure (B) The Welch T test was used to compare the two groups. The nodule represent the root nodules of different alders, soil represent the associated soils of three alders, respectively. Asterisks indicate significant differences between samples, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

LDA effect size (LEfSe) analysis of the A mandshurica (A), A sibirica (B), and A japonica (C) microbial communities with an LDA score of > 4.0 (p < 0.05). Phylogenetic levels from phylum to species are represented by circles. Different-colored nodes represent microbial groups that are significantly enriched in the corresponding groups and have a significant influence on the differences between groups. The yellow nodes represent microbial groups that have no significant difference between groups or have no significant effect on the differences between groups.

Figure 5

Interaction network of dominant microbiota at the genus level (top 30) in root nodules and associated soils. (A) Interaction network in root nodule. (B) Interaction network in root nodule surface soils. The size of the nodes shows the abundance of the genus, and the different colors indicate the corresponding taxonomic assignment at the phylum level. The edge color represents positive (red) and negative (green) correlations. The edge thickness indicates the correlation values; only show significant interactions are shown (|r| > 0.6; p < 0.05).

Figure 6

Comparison analysis of the abundance of the microbial nitrogen cycling pathways among different nodule and soil samples. (A) The differential abundance of the microbial nitrogen cycling pathways between nodules and soils. (B) The differential abundance of the microbial nitrogen cycling pathways in root nodules of A sibirica from five different eco-geographical environments. The Welch T test and one-way ANOVA were used to compare multiple groups. Asterisks indicate significant differences among groups, *p < 0.05, **p < 0.01, ***p < 0.001.