The ability of C. violifolia assimilating different types of Se
The experiments were designed to examine the ability of C. violifolia utilizing Se and soil microbiome (Table 1). Four types of Se sources with different concentrations (0, 50, 100, 200, 400, and 800 mg/L) were added into the soils, and the Se concentration in both the soils and C. violifolia was compared in different treatments. Consistent with our previous work (Wu et al. 2020), among these different selenium sources, selenate was the best for increasing the Se concentration in the leaves of C. violifolia, following by selenite, B. subtilis-Se, and yeast-Se in the concentration rang tested. The concentration of Se in the leaves of C. violifolia was positively related to the supplementation of B. subtilis-Se, selenite, and selenate into the soils. However, the addition of yeast-Se in the soil was not linearly related to the Se concentration in the leaves of C. violifolia.
To quantified by the ratio of Se concentrations in C. violifolia to that in the soils, the Se uptake of plant from soil was investigated in different treatments. When there was no supplementation of exogenous Se, the concentration of Se in the soil was 1.54 ± 0.4 mg/kg. The addition of these four types of Se increased the Se concentration in the soils. However, only the uptake of the selenate group had a higher ratio than the plant control group (C. violifolia without Se addition) (Fig. 1). In addition, the absorption ratio was not decreased when the concentration of exogenous selenate was increased. These data suggested that unknown mechanism existed in C. violifolia for actively assimilating selenate. The absorption ratios of other three types of Se sources were lower than the plant control. The ratio was reduced in higher concentrations of yeast-se and selenite and fluctuated for B. subtilis-Se, indicating that C. violifolia could not utilize high concentration of yeast-Se, selenite, B. subtilis-Se or as well as selenate. A large part of extra yeast-Se, selenite, and B. subtilis-Se could not be absorbed by C. violifolia and they might be kept in the soils or volatilize, which might be beneficial for C. violifolia growth.
Se is indispensable for humans, but it is not clear if the trace element Se is indispensable for plants growth. Many studies have indeed found that Se showed beneficial effects for various plants. It can promote growth, increase tolerance to heavy metals, confer resistance to pathogens, and protect from oxidative damage (Djanaguiraman et al. 2010; Pandey and Gupta 2015; Schiavon and Pilon-Smits 2016). However, the beneficial effects of Se are usually identified at low level, and high concentration of exogenous Se application inhibited plant growth or lead to Se toxicity occurrence (Hui et al. 2011). This is consistent with our results. In addition, selenate showed the highest accumulation in C. violifolia among the different forms of Se. This might be because selenate was more readily assimilated through the transporter in the root cell membrane (Wu et al. 2020). Therefore, the source and concentration of Se should be taken into attention when exogenous Se fertilizers are applied.
The influence of Se sources on the diversity of C. violifolia rhizosphere microbiome
Rhizospheremicrobiota has been reported to help plant growth via the acquisition of nutrients or the prevention against harmful substance in soil (Mendes et al. 2013). To investigate which rhizosphere microbes could promote the Se enrichment in C. violifolia, the first thing is to characterize the impact of Se to the rhizosphere microbiome of C. violifolia. The microbial profiles were compared under different exogenous Se treatments (Table 1). We would like to identify the bacteria species with increased abundance in higher concentration of exogenous Se.
The alpha rarefaction plotting of Shannon/OTU suggested that the sequencing depth was enough to cover most of the species in the rhizosphere microbiome. Alpha diversity was performed using three index, Shannon index, observed OTU, and evenness. The related differences were analyzed using Kruskal–Wallis test. Most of the Se treatments could not affect the Shannon diversity in the rhizosphere microbiome of C. violifolia except fromthe two samples 800 mg/mL B. subtilis-Se and 200 mg/mL yeast-Se (Fig. 2a). Additionally, the different alpha diversities in these two sets of microbiomes were caused by both the changes of OTU numbers and microbial evenness (Fig. 2b and c). The supplementation of B. subtilis-Se reduced the observed OTUs at the levels of 100, 400 and 800 mg/mL and decreased evenness and Shannon index at 800 mg/mL level (Fig. 2b and c), indicating that some rhizosphere microbes could be inhibited or killed by B. subtilis-Se. In the treatment of 200 mg/mL yeast-Se, more species were observed and the microcial abundances were more uniformly distributed (Fig. 2b and c). However, higher concentrations of yeast-Se reduced the alpha diversity to the level of the control group. One explanation was that low concentration of yeast-Se (≤ 200 mg/L) might contain nutrient that helped the growth of some rhizosphere microbiota, but high concentration of yeast-Se (≥ 400 mg/L) might inhibit the survival of the rhizosphere microbes. Because both the B. subtilis and yeast-Se were not purified Se compounds, the impact of them on the rhizosphere microbiome might be induced by other components in the Se sources. Nevertheless, the influence from B. subtilis- and yeast-Se to the root microbiome should be involved in the observed changes.
Beta diversity was quantified using Bray-Curtis distance and the difference between each sample and the plant control was calculated by Mann-Whitney U test (Fig. 3a). Furthermore, ordination analysis was performed via non-metric multidimensional scaling (NMDS) (Fig. 3b). The root microbiome was more dissimilar to the plant control with the supplementation of B. subtilis-Se, suggested the impact of B. subtilis-Se on the rhizosphere microbiome of C. violifolia was in a concentration-dependent manner (Fig. 3). This phenomenon was consistent with the reduced alpha diversity by B. subtilis-Se treatment, which might because some microorganisms were inhibited or killed by exogenous B. subtilis-Se (Fig. 2). Although selenate was the favorite Se source for C. violifolia (Fig. 1), the alpha diversity of the rhizosphere microbiome was not affected by selenate (Fig. 2), and the beta diversity was only affected by 50 mg/mL of selenite (Fig. 3). A reasonable explanation was that most of selenate was assimilated by C. violifolia, and as a result, selenate had a low concentration in the soil (Fig. 1b), which might limit its influence on the rhizosphere microbiome. As such, selenate was the closest group to the plant control in NMDS figure (Fig. 3b). The beta diversity of no plant was significantly different from plant control (Fig. 3), suggesting that C. violifolia affected soil microbiome. A significantly lower evenness of the microbiome in plant control samples than that in no plant samples illustrated that the existence of C. violifolia might enrich some microorganisms in the soil (Fig. 2).
Species affected by Se sources in the rhizosphere microbiome of C. violifolia
We trained a Qiime2 feature classifier using SILVA database for the identification of species in the rhizospheremicrobiome of C. violifolia. Top five phylum in the rhizosphere microbiome of C. violifolia were Actinobacteria (42.3%), Proteobacteria (32.4%), Bacteroidetes (7.1%), Firmicutes (3.8%) and Patescibacteria (3.7%) (Fig. 4). The rhizosphere microbiome profiles of four Se were compared to plant control at phylum level using Mann-Whitely U test. The application of B. subtilis- and yeast-Se slightly changed bacterial abundance at the phylum level compared to selenate and selenite (Fig.6). B. subtilis-Se application enriched the abundance of Deinococcus-Thermus, Dependentiae, and Patescibacteria, and reduced that of Epsilonbacteraeota, Elusimicrobia, Chlamydiae,and Acidobacteria. Yeast-Se application enriched the abundance of Patescibacteria, Acinobacteria, and Firmicutes, and reduced that of Epsilonbacteraeota, Spirochaetes, Elusimicrobia, Chlamydiae, and Acidobacteria. The change profile was similar between selenate and selenite application, and they both increased Deinococcus-Thermus, Halanaerobiaeota, Acitnobacteria, Patescibacteria, and Dependentiae, and reduced Epsilonbacteriaeota, Spirochaetes, Verrucomicrobia, Chlamydiae, and Acidobacteria. The only difference was that selenite also increased Firmicutes and reduced Elusimicrobia (Fig. 5).
Although the most exact species were not identified, some genera differences were found between the rhizosphere microbiome profiles of four Se application and plant control. B. subtilis-Se application changed the most types of microbes, and it enriched the abundance of Leucobacter, Sporosarcina, Patulibacter, and Denitrobacter and reduced that of Mycobacterium. Yeast-Se application enriched the abundance of Singulishaera, Lactobacillus, Bdellovirio, and Bosea. Selenite sample enriched only Bosea, andselenate sample was found to enrich only Soirubrobacterales at the order level. Furthermore, theabundance increase of Solirubrobacterales and Bosea was, respectively, positively related to the concentration of selenate and selenite application as quantified by linear regression (Fig. 6b and c).
Leucobacter was reported to be tolerant for high heavy metals, such as chromate, and therefore it was used for heavy metal reduction (Zhu et al. 2008). Another previous study found that B. subtilis application increased Leucobacter abundance (Ding et al. 2021), indicating that some components or metabolites from B. subtilis is benefitable for Leucobacter growth. In our result B. subtilis-Se application showed the biggest abundance increase for Leucobacter, indicating that Leucobacter might be tolerant for high concentration of Se. Some species which belong to Singulishaera were often identified in acidic environment (Kulichevskaya et al. 2008; Kulichevskaya et al. 2012), a similar condition with yeast’s growth environment. Although the Yeast-Se is inactive, it is speculated that some organic compounds from yeast might be benefitable for Singulishaera enrichment. Solirubrobacterales was ubiquitous in natural environments, including rock, cropland, and lake (Khilyas et al. 2019; Liu et al. 2020), and it was also found to be enriched in in the high-selenium-contaminated reclaimed mine soils (Rosenfeld et al. 2018), which was consistent with our results. Some reports showed that Bosea was present in the rhizosphere or root nodules of plants (Qaisrani et al. 2019; Safronova et al. 2020; Sazanova et al. 2019), and itwas also frequently found to perform denitrification function or play antioxidant protective roles (Hassan et al. 2019; Tian and Wang 2021; Wang et al. 2020; Zhang et al. 2021). These results indicated that Bosea might use selenite as electron receptor and play key roles during the selenite reduce in the rhizosphere of C. violifolia. Taken together, these results showed that various Se source application shaped the different rhizosphere microbiome profiles. Reversely, these might affect Se uptake and transform of C. violifolia, which is worthy of future investigation.