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 ).