Carbon nanosol-induced assemblage of a plant-beneficial microbiome consortium

Abstract

Carbon nanosol (CNS) is a carbon-based nanomaterial that promotes plant growth; however, its functional mechanisms and effects on the microbiome are not fully understood. Here, we explored the effects of CNS on the relationship between the soil, endophytic microbiomes and plant productivity. CNS treatment increased the fresh biomass of tobacco (Nicotiana tabacum L.) plants by 27.4% ± 9.9%. Amplicon sequencing analysis showed that the CNS treatment significantly affected the composition and diversity of the microbial communities in multiple ecological niches associated with tobacco, especially the bulk soil and stem endophytic microbiome. Furthermore, the application of CNS resulted in enhanced network connectivity and stability of the microbial communities in different niches, particularly in the soil, implying a strengthening of certain microbial interactions. Certain potentially growth-promoting root endophytic bacteria were more abundant under the CNS treatment. In addition, CNS increased the abundance of some endophytic microbial functional genes known to enhance plant growth, such as those associated with nutrient metabolism and the plant hormone biosynthesis pathways. We isolated two bacterial strains (Sphingopyxis sp. and Novosphingobium sp.) that were enriched under CNS treatment, and they were confirmed to promote tobacco plant growth in vitro. These results suggested that CNS might, at least in part, promote plant growth by enriching beneficial bacteria in the microbiome.

Keywords: Carbon nanosol; Microbiome; Nano biofertilizer; PGPR; Plant growth; Sustainable agriculture.

Fig. 1

Effects of CNS on the growth of tobacco in a pot experiment. A Morphology of tobacco after 15 days of applying CNS at concentrations of 15 µg/mL three times to the soil in a pot experiment. B Effects of CNS on tobacco growth indicators. Asterisks denote significant differences (t test, P ≤ 0.05) between the CNS-treated and control (CK) samples treated with water, and two asterisks indicate P < 0.01

Fig. 2

Diversity of tobacco-associated microbiomes. A Nonmetric multidimensional scaling (NMDS) ordinations based on weighted UniFrac distance matrices depicting the distribution patterns of bacterial and fungal communities in each compartment niche (for each niche, n = 12). The relative contribution of different factors to community dissimilarity was tested with PERMANOVA. “T” represents the effect of the CNS treatment. B Alpha-diversity of bacterial and fungal communities in soils (rhizosphere and bulk soils) and plant compartments (root endosphere and stem endosphere). Asterisks (“*”, P < 0.05; “**”, P < 0.01; “***”, P < 0.001) above the boxes indicate a significant difference between control and treatment within a compartment, determined using a nonparametric Kruskal–Wallis test

Fig. 3

Community composition of soil- and plant-associated microbiomes. A Relative abundance of bacterial and fungal communities at the phylum level in the CNS-treated and control (CK) samples for four compartments: bulk soils (BS), rhizosphere (RS), root endosphere (R), and stem endosphere (S). B Fold change of bacterial and fungal genera related to plant growth–promoting ecological functions that were significantly affected by CNS

Fig. 4

Microbial interkingdom networks within each niche. Co-occurrence network analysis showing microbial interkingdom network patterns differed clearly for CNS-treated and control (CK) samples in each plant niche; ave.d means the average degree of all nodes

Fig. 5

Functional profiles of tobacco microbiomes. A Nonmetric multidimensional scaling (NMDS) ordination analysis based on Bray–Curtis distance matrices of the KEGG ontology annotations, showing that the CNS-induced tobacco microbiome significantly differed from the control microbiome (n = 48). B Boxplot of the functional diversity of microbiomes in four compartments. Asterisks above the boxes indicate a significant difference (P < 0.01). C Heatmap exhibiting the relative abundance of functional genes associated with plant growth–promotion functions. All genes associated with plant growth–promotion functions are listed in Additional file 2: Table S5. The four compartments are bulk soils (BS), rhizosphere (RS), root endosphere (R), and stem endosphere (S)

Fig. 6

The effect of two isolated bacteria on tobacco growth. A Phenotype of tobacco plants after inoculation with two bacteria on MS plates. B Effects of single inoculation with B-25 (Sphingopyxis sp.) or B-29 (Novosphingobium sp.) and coinoculation of two strains (B25 + B29) on different tobacco growth parameters. ANOVA with an LSD test (p < 0.05) indicated statistically significant differences denoted by different letters for each assessed parameter. C Phylogenetic analysis of isolated bacteria. Neighbor-joining trees were constructed using partial 16S rRNA sequences of the two strains and their close relatives. Escherichia coli was used as an outgroup for rooting the tree