What If Root Nodules Are a Guesthouse for a Microbiome? The Case Study of Acacia longifolia

Abstract

Acacia longifolia is one of the most aggressive invaders worldwide whose invasion is potentiated after a fire, a common perturbation in Mediterranean climates. As a legume, this species establishes symbioses with nitrogen-fixing bacteria inside root nodules; however, the overall microbial diversity is still unclear. In this study, we addressed root nodules' structure and biodiversity through histology and Next-Generation Sequencing, targeting 16S and 25S-28S rDNA genes for bacteria and fungi, respectively. We wanted to evaluate the effect of fire in root nodules from 1-year-old saplings, by comparing unburnt and burnt sites. We found that although having the same general structure, after a fire event, nodules had a higher number of infected cells and greater starch accumulation. Starch accumulated in uninfected cells can be a possible carbon source for the microbiota. Regarding diversity, Bradyrhizobium was dominant in both sites (ca. 77%), suggesting it is the preferential partner, followed by Tardiphaga (ca. 9%), a non-rhizobial Alphaproteobacteria, and Synechococcus, a cyanobacteria (ca. 5%). However, at the burnt site, additional N-fixing bacteria were included in the top 10 genera, highlighting the importance of this process. Major differences were found in the mycobiome, which was diverse in both sites and included genera mostly described as plant endophytes. Coniochaeta was dominant in nodules from the burnt site (69%), suggesting its role as a facilitator of symbiotic associations. We highlight the presence of a large bacterial and fungal community in nodules, suggesting nodulation is not restricted to nitrogen fixation. Thus, this microbiome can be involved in facilitating A. longifolia invasive success.

Keywords: Bradyrhizobium; Coniochaeta; NGS; fire event; histology; infected cells; starch; symbiosis.

Figure 1

Longitudinal semi-thin sections (12 µm) of Acacia longifolia root nodules collected in unburnt and burnt sites. Nodules have four different zones: meristematic zone with a persistent meristem (M) or zone I, infection zone (IZ) or zone II, N-fixing zone (NFZ) or zone III, and senescence zone (SZ) or zone IV. Vascular bundles (VB) are present in the nodule cortex (C). The central infected zone contains infected (IC) and uninfected (UC) cells, and starch granules (S) are present in UC in close vicinity to IC. Senescent cells (SC) were observed in SZ. Reconstructed images on the left were stained with toluidine blue. Magnified images on the right side refer to three different insets: infection zone (identified as ZII), nitrogen-fixing zone (ZIII), and senescent zone (ZIV). Insets (a,b,e) are stained with cotton blue in lactophenol, (c,f) are unstained, (d,h) are stained with Lugol solution, and (g) is stained with Löffler solution. (*) represents rod-shape bacteria inside IC and arrows highlight the quantity of IC. Note that scale bars are different and adjusted to each image.

Figure 2

Top 10 genera of the bacteria identified in Acacia longifolia root nodules (cumulative relative abundance, %), for unburnt and burnt sites. The bottom graph represents an inset of the abundance excluding Bradyrhizobium (23%). The percentage was calculated based on the mean value of reads assessed for each site (n = 3) and is presented in the legend considering unburnt followed by burnt values. Colors are assigned according to bacterial classes (gradient of purple, Alphaproteobacteria; gradient of green, Betaproteobacteria; gradient of orange for Cyanophyceae; blue for Mollicutes).

Figure 3

Top 10 genera of the fungal community identified in Acacia longifolia root nodules (cumulative relative abundance, %), for unburnt and burnt sites. The percentage was calculated based on the mean value of reads assessed for each site (n = 3) and are presented in the legend considering unburnt followed by burnt values. Colors are assigned according to fungal phyla (gradient of green, Ascomycota; gradient of blue, Basidiomycota; grey for Mucoromycota).