Disentangling the influence of earthworms in sugarcane rhizosphere

For the last 150 years many studies have shown the importance of earthworms for plant growth, but the exact mechanisms involved in the process are still poorly understood. Many important functions required for plant growth can be performed by soil microbes in the rhizosphere. To investigate earthworm influence on the rhizosphere microbial community, we performed a macrocosm experiment with and without Pontoscolex corethrurus (EW+ and EW−, respectively) and followed various soil and rhizosphere processes for 217 days with sugarcane. In EW+ treatments, N2O concentrations belowground (15 cm depth) and relative abundances of nitrous oxide genes (nosZ) were higher in bulk soil and rhizosphere, suggesting that soil microbes were able to consume earthworm-induced N2O. Shotgun sequencing (total DNA) revealed that around 70 microbial functions in bulk soil and rhizosphere differed between EW+ and EW− treatments. Overall, genes indicative of biosynthetic pathways and cell proliferation processes were enriched in EW+ treatments, suggesting a positive influence of worms. In EW+ rhizosphere, functions associated with plant-microbe symbiosis were enriched relative to EW− rhizosphere. Ecological networks inferred from the datasets revealed decreased niche diversification and increased keystone functions as an earthworm-derived effect. Plant biomass was improved in EW+ and worm population proliferated.

The values for K, Ca, Mg, Al, H+Al, SEB and CEC are represented according the following unit: mmolc.dm-3; for all the others except pH, the unit represented is: mg.dm-3, pH unit is CaCl2 0.01 mol/L. All the variables were statistically tested following the methods as described in the main text. No significant differences were detected.
Figure S1 | Rarefaction curves obtained from the functional profiling of the metagenomic datasets in bulk (a) and rhizosphere (b). The curves indicate the coverage of microbial functions assigned to the INTERPRO2GO database for each dataset. The read counts were normalized to the smallest number of reads (MEGAN6). Each leave represents a different function assigned within the tree of functions obtained from MEGAN6. The lines in the plots represent each one of the 12 metagenomic samples obtained at the 217th day after the beginning of the experiment.   Figure S2 | Microbial functions altered in bulk (a) and in rhizosphere (b). The error bar plots indicates the p-value of the functions with the effect size and associated confidence interval for each function detected to be of significant biological relevance (t-test, p-value<0.05). The color code indicated in the confidence intervals shows if the enrichment was higher in EW-(blue) or in EW+ (orange).  * Measured according to the number of shortest paths between any two nodes that pass through one particular node. High values indicate high influence of the node on the model.

Microcosm experiment
Plastic pots (5L) were filled with sieved and homogenized soil collected from the same location as was for the macrocosm experiment (University of Sao Paulo -Experimental Station, Piracicaba) and incubated in the greenhouse for 30 days with and without sugarcane seedlings and with and without earthworms (n=4). The earthworms from the same specie (P. corethrurus) were obtained from a specialized producer (Minhobox) and followed the same procedure of pre-incubation as described for the macrocosm experiment. Two sugarcane seedlings at the same developmental stage were planted per pot and 20 individuals of young earthworms were inoculated per pot. For sampling the gas belowground it was used the same method described for the macrocosm experiment with airstones buried (15 cm depth) in the center of pots.
After 30 days gas samples belowground were collected and a destructive sampling was performed, and rhizospheric soil and earthworms were incubated for gas measurements. 1 gram (fresh weight) of rhizospheric soil (n=3) from the pots with earthworms (EW+) and without it (EW-), or 1 individual of earthworm (n=3) from the pots with sugarcane (SC+) and without it (SC-) was incubated for 5 minutes inside 10 ml syringes in the dark at room temperature. Prior to the incubation, worms were washed in sterilized water, dried with a paper towel and transferred to the 10 ml syringe. After transferring the rhizospheric soil or the earthworm, the volume of the syringe was set to 10 ml and the plastic cap (replacing the needle) was closed, so that there was no variation in the pressure and the atmosphere inside was not exchanged with the outside. After incubation time the syringe was connected to the chromatograph, the plastic cap of the syringe was opened and a sample of air was injected. After injection of the syringe air into the chromatograph, worms were removed from the syringes and weighted (0.29 g ±0.07). Therefore the gas emission from earthworms was normalized per gram of the individual by dividing the concentrations obtained by the fresh weight of the worm incubated. The variables measured were tested statistically for homogeneity and normal distribution prior to the identification of appropriate statistical tests for detecting differences between the means.

Gas emissions from the incubations
The N 2 O emissions (Supplementary Figure S3a) detected belowground were different (Kruskal-Wallis, p-value=0.01) among the treatments. N 2 O emissions from the pots with only sugarcane (EW-SC+) were not different from the emissions of the pots with only soil (EW-SC-) (Dunn's test. p-value=0.60). However, the N 2 O emissions from the pots with earthworms were significantly higher compared to the emissions of the pots without worms (i.e., EW+SC-compared to EW-SC-, Dunn's test, p-value=0.009346;and EW+SC-compared to EW-SC+, Dunn's test, p-value=0.03759;and EW+SC+ compared to EW-SC-, Dunn's test, p-value=0.01157;and EW+SC+ compared to EW-SC+, Dunn's test, p-value=0.04496). Although, the N 2 O emissions between the pots with worms were not different (i.e., EW+SCcompared to EW+SC+,Dunn's test,).
Differences were also detected for the CO 2 emissions (Supplementary Figure  S3d) from belowground (Kruskal-Wallis test, p-value=0.008). CO 2 was higher in all the pots with earthworms or with sugarcane (i.e., EW-SC+ compared to Dunn's test,Dunn's test,Dunn's test,, and no significant difference were detected between them (Dunn's test, p-value>0.05). N 2 O and CO 2 emissions of rhizospheres, (Supplementary Figure S3b, e), were not different (t-test, p-value>0.05). In vivo emissions of worms (Supplementary Figure S3c, f) were not different for N 2 O (t-test, p-value>0.05), but CO 2 emissions of worms from pots with sugarcane were significantly higher compared to the pots without plants (ttest, p-value=0.00929). Further, in vivo emissions of N 2 O of P. corethrurus were significantly higher than the N 2 O emissions of rhizospheric soil from the pots with earthworms (t-test, p-value=2.5x10 -6 ).
Figure S3 | Verification study: N 2 O and CO 2 emissions belowground, from rhizospheric soils, and earthworms. Panels a), b) and c) show N 2 O concentrations (mM). Panels d), e) and f) show CO 2 concentrations (mM). Panels a) and d) show the gas concentrations belowground at the end of the experiment (day 30 th ) for the treatments (n=4): without earthworms and sugarcane (EW-SC-), with sugarcane (EW-SC+), with earthworms (EW+SC-), and with both (EW+SC+). Panels b) and e) show the mean concentration of gas emitted of rhizospheric soils (1 gram of fresh weight) from the pots with earthworms (EW+) and without it (EW-). The panels c) and f) show the in vivo gas emissions per gram (fresh weight) of earthworm from the pots with sugarcane (SC+) and without it (SC-). Different letters above the boxes in the panels indicate significant differences (p<0.05).