Root exudation
To evaluate the possible mechanisms determining root-associated microbiomes via chickpea genetic variation and longitudinal niche differentiation, we analysed root exudation from apical and basal segments of chickpea genotypes with genetic variation in flowering time. To remove the microbial effect in degrading root exudates, sterilised soil (Soil-C) was used in the same rhizotron system and growing conditions as the microbiome experiment. Primary metabolites were analysed from the collected rhizosphere soil and adjusted by their concentrations in bulk soil. Based on the 34 detected root exudate compounds, unconstrained principal coordinate analyses (PCoA) and PERMANOVA using Bray-Curtis distance were performed, both of which demonstrated the considerable effect of longitudinal root segment on root exudate composition, indicated by high R2 value of 57% (Table S2). In the biplot, a separation was observed between samples of apical root and basal root, with the PCoA1 explaining 55% of the variation (Fig. 5a). In the apical root, the root exudate composition from the EF genotype differed greatly from the LF genotypes, while for the compounds from basal root, genotype LF2 differed from the other two genotypes.
The total amount of detected compounds from root exudation was greater in the root apical zone than the basal zone (Table S1). For the apical root, the EF genotype excreted more compounds than the LF genotypes; in particular, carboxylates from EF were 53% to 88% greater than those from the LF genotypes (Fig. 5b). There was no genotypic variation in rhizosphere soil pH (Table S2), and the rhizosphere soil at the apical zone had a lower pH than the basal zone (4.30 vs 4.85, P < 0.01, Fig. 5c). The change of rhizosphere soil pH was associated with the abundance of certain root exudate compounds, in particular, the amount of total carboxylates was negatively correlated with rhizosphere soil pH (Table S3).
For every root exudate detected, we compared its abundance in the EF genotype with each of the LF genotypes, and selected the compounds with Log2 fold changes (FC) to both LF genotypes > 1 and FDR-adjusted P < 0.01 as EF-enriched, and Log2 FC < -1 and FDR-adjusted P < 0.01 as LF-enriched (Fig. 5d). No EF- or LF- enriched root exudates were found in the basal zone, while in the apical zone, propionic acid (carboxylate), benzoic acid (carboxylate), and phenylalanine (carboxylate) were identified as EF-enriched compounds, and succinic acid (carboxylate), mannose (sugar) and trehalose (sugar) were LF-enriched root exudates. The abundance of the three EF-enriched root exudates were highly correlated with the relative abundance of three core ASVs in apical root (Table S4) including ASV07563 (unclassified Burkholderiaceae), ASV25120 (Rhizobacter ), and ASV30169 (Methylotenera ).