Mucilage produced by aerial roots hosts diazotrophs that provide nitrogen in Sorghum bicolor

Abstract

Sorghum (Sorghum bicolor) is an important food, feed, and fodder crop worldwide and is gaining popularity as an energy crop due to its high potential for biomass production. Some sorghum accessions develop many aerial roots and produce an abundant carbohydrate-rich mucilage after rain. This aerial root mucilage is similar to that observed in landraces of maize (Zea mays) from southern Mexico, which have been previously shown to host diazotrophs. In this study, we characterized the aerial root development of several sorghum accessions and the impact of humidity on this trait. We conducted a microbiome study of the aerial root mucilage of maize and sorghum and isolated numerous diazotrophs from field sorghum mucilage. We observed that the prevailing phyla in the mucilage were Pseudomonadota, Bacteroidota, and Bacillota. However, bacterial abundances varied based on the genotype and the location. Using acetylene reduction, 15N2 gas feeding, and 15N isotope dilution assays, we confirmed that these sorghum accessions can acquire about 40% of their nitrogen from the atmosphere through these associations on aerial roots. Nitrogen fixation in sorghum aerial root mucilage offers a promising avenue to reduce reliance on synthetic fertilizers and promote sustainable agricultural practices for food, feed, fodder, and bioenergy production.

Fig 1. Sorghum screening of selected accessions under greenhouse conditions.

Different sorghum accessions display diverse traits associated with aerial roots and mucilage production. (A) Number of nodes in different sorghum accessions (n = 3–6). Most of the accessions evaluated showed more than five nodes with aerial roots. (B) Representative image of selected sorghum accessions. Scale bar = 28 cm. (C) Volume of mucilage per root (μl) produced by different sorghum accessions (n = 4–9). The production of mucilage varied among all the accessions. (D) Diameter (mm) of aerial roots in different sorghum accessions (n = 6–15). Thick roots with diameters greater than 4 mm were noted in nearly all the accessions. (E) Aerial roots of the sorghum accessions in B. Scale bar = 28 cm. An ANOVA (F-test) to estimate significant differences between means was conducted with the package multcompView (0.1–10). The data used to generate the plots is available in S2 Dataset.

Fig 2. Effect of humidity in sorghum accession IS23992 and IS24453.

Boxplots of different phenotypes measured in two sorghum accessions under high (75%) and low (30%) relative humidity. (A) Number of nodes with aerial roots (n = 8). The number of nodes with aerial roots significantly increased in both accessions. (B) Number of roots on the top node (n = 8). The number of roots at the top node only increased for IS24453. C) Average root diameter on the top node (n = 8). In neither of the accessions was the root diameter affected by the humidity. A Wilcoxon test was performed in R (Ver 4.2.1) with the package ggpubr (Ver 0.6.0) to determine the effect of the humidity on the aerial root phenotypes. * denotes p ≤ 0.05, ** denotes p ≤ 0.01, and n.s., not significant. The data used to generate the plots is available in S2 Dataset.

Fig 3. Sorghum aerial root mucilage composition.

Sorghum aerial roots release mucilage. (A) Mucilage production is triggered by water and under high-humidity conditions. The mucilage from different accessions appears similar. Accession IS23992 serves as a representative example of sorghum mucilage. Scale bar (5 cm). (B) Monosaccharide composition of sorghum accession IS11026 and Sierra Mixe maize mucilage. Both mucilages contain the same monosaccharides but in different proportions (mole %). (C) Concentration of reducing sugars in sorghum mucilage from the accession IS23992 determined with Benedict’s reaction. A Wilcoxon test was performed to compare means at the two-time points. *** denotes p ≤ 0.001 (n = 8). (D) Principal component analysis using Sparse Partial Least Squares. Two clusters are depicted based on the species studied: sorghum accessions IS29091 (green diamonds) and IS2245 (green circles) and maize landraces CB017456 (red triangles) and GRIN 19897 (red circles). The data used to generate the plots is available in S2 Dataset.

Fig 4. Microbiome profile of sorghum mucilage.

The community profile shows the relative abundance of bacteria present in sorghum (S) and maize (M) mucilages. Samples S1 to S4 (squares) and M1 to M6 (triangles) were collected from the field at the University of Wisconsin-Madison (UW-Madison), and samples S5 to S7 (circles) were collected at the University of Florida (UF). (A) Principal coordinates analysis plot of sorghum and maize samples based on 16S amplicon sequences. (B) Relative abundance of sorghum and maize samples based on 16S amplicons and OTUs. (C) Unrooted maximum-likelihood phylogenetic tree of 103 bacteria isolated from sorghum aerial roots mucilage in BMGM nitrogen-free semi-solid medium. The evolutionary distances were computed using the Jukes–Cantor method and are in the units of the number of base substitutions per site. This analysis involved 103 nucleotide sequences. All positions containing gaps and missing data were eliminated (complete deletion). There were 908 positions in the final dataset. Numbers at nodes are bootstrap values (1,000 replications, values <50% are not shown). Colors: Red: Gammaproteobacteria, Purple: Betaproteobacteria, Blue: Alphaproteobacteria, Brown: Bacillota, Green: Actinomycetota, Yellow: Bacteroidota. (D) Nitrogenase activity of 34 isolated from sorghum aerial roots mucilage in BMGM nitrogen-free semisolid medium (n = 3). The red arrows show the reference strains for comparison. Bacteria: Klebsiella variicola A3 ΔnifH. Species codes (alphabetic order): Agrobacterium divergens, Agrobacterium fabaceae, Agrobacterium larrymoorei, Agrobacterium pusense, Azospirillum brasilense, Azospirillum humicireducens, Azospirillum palustre, Bacillus amyloliquefaciens, Enterobacter cancerogenus, Epilithonimonas hungarica, Herbaspirillum seropedicae, Klebsiella michiganensis, Klebsiella oxytoca, Klebsiella variicola, Microbacterium aerolatum, Microbacterium binotii, Microbacterium hominis, Microbacterium neimengense, Microbacterium oleivorans, Microbacterium testaceum, Novosphingobium kaempferiae, Phytobacter diazotrophicus, Pseudoacidovorax intermedius, Pseudomonas bharatica, Pseudomonas campi, Pseudomonas lutea, Pseudomonas sediminis, Pseudomonas turukhanskensis, Pseudoxanthomonas winnipegensis, Rhizobium cellulosilyticum, Siphonobacter intestinalis, Stenotrophomonas lactitubi, Stenotrophomonas nematodicola, Stenotrophomonas rhizophila, and Stenotrophomonas terrae. Mucilage from the sorghum accessions were the same as described in Wolf and colleagues [28]. The data used to generate the plots is available in S2 Dataset.

Fig 5. Biological nitrogen fixation on aerial roots from sorghum.

(A) Acetylene reduction assay in mucilage generated in the greenhouse. Mucilage was used as medium to grow nine nitrogen-fixing strains (Azospirillum brasilense FP2, Azospirillum baldaniorum, Azorhizobium caulinodans, Azotobacter vinelandii DJ, Herbaspirillum seropedicae, Klebsiella michiganensis, Klebsiella variicola A3, Paraburkholderia silvatlantica, and Pseudomonas stutzeri) and Azospirillum brasilense FP10 as a non-fixing control. Tukey's honestly significant difference (HSD) test was performed in R, and significant group means are displayed with lowercase letters (n = 6). (B) Analysis of ¹⁵N gas feeding experiment on aerial roots of sorghum accession IS23992. Aerial roots were inoculated with K. variicola ΔnifH (negative control) or K. variicola A3 (positive control). *** indicates p ≤ 0.001 based on Wilcoxon signed-rank test (n = 8). (C) ¹⁵N isotope dilution experiment on aerial roots of sorghum accessions IS23992 and IS24453. These accessions were inoculated with the nine strains from the ARA experiment (diazotrophs), and the three non-fixing mutant strains Azospirillum brasilense FP10, Azotobacter vinelandii DJ100 ΔnifH, and Azorhizobium caulinodans ORS571 ΔnifA. Whole plants at the flowering stage were ground, and the average of three replicates was plotted. Percent Ndfa was calculated for both accessions using the plants inoculated with diazotrophs and using the plants inoculated with mutant strains as a reference. An ANOVA was performed to compare means between treatments and genotypes using the package multcompView. (D) Scatterplot of the percentage of nitrogen derived from the atmosphere (%Ndfa) in sorghum samples collected at different developmental stages from the accessions IS23992 and IS24453, indicated with circles and triangles, respectively. Solid and dotted lines are regression lines for IS23992 and IS24453, respectively. Samples were collected every 2 weeks, starting at stage 3 (Samples S3 to S5) and after flowering (S6). Error bars display standard deviations. Pair-wise comparisons were made with Wilcoxon’s test;**** denotes p ≤ 0.0001 and n.s., not significant. The data used to generate the plots is available in S2 Dataset.