3.3 Functional potential of the rhizosphere microbial community
Bacterial functional profiles were inferred from 16S rRNA gene data (Appendix S3) using PICRUSt. Differences between the control and test groups were observed with a strong segregation (p < 0.05), including the notable enrichment of numerous pathways including peptidases, amino acid metabolism, starch and sucrose metabolism, porphyrin and chlorophyll metabolism, carbon fixation, flagellar assembly, and glycosyltransferases (Figure 5a, Appendix S4). Biological functions were found significantly abundant in the SPF treatment compared with those in the SK treatment (p < 0.05), including lipid metabolism, cell motility, metabolism of amino acids, glycan biosynthesis, and xenobiotics biodegradation. Through comparisons of the bacterial function in groups SPF and SP using t-test, we found that SPF exhibited significantly higher xenobiotics biodegradation and metabolism (p < 0.05) (Appendix Figure S5a). SPF had significantly increased functions for metabolism of cofactors and vitamins compared to SF (p < 0.05) (Appendix Figure S5b), as well as increased functions of metabolism compared to SA (p < 0.05) (Appendix Figure S5c). As the only different compound between the SP and SF groups was clothianidin, the distinctive functions caused by clothianidin mainly included pyruvate metabolism, glycolysis, carbon fixation, butanoate metabolism, and methane metabolism, which were significantly higher than that in the SF treatment (p < 0.05) (Figure 5a). This results demonstrates that these functions would be positively involved in the transformation and degradation of clothianidin in soil.
We allocated 1494 total fungal OTUs (Appendix S5) to fungal functional groups using the FUNGuild annotation tool. At least eight trophic modes were detected in this study, among which the saprotroph mode was the most abundant, followed by pathotroph, pathotroph-saprotroph, pathotroph-saprotroph-symbiotroph, and symbiotroph (Figure 5b, Appendix S6). Sequences annotated with the saprotroph, pathotroph-saprotroph and pathotroph-symbiotroph modes were significantly decreased in the SPF group (p < 0.05) than those in the SK and SA groups. At the guild level, the most abundant guilds were saprotrophs, followed by plant pathogens, soil saprotrophs, and wood saprotrophs (Figure 5c, Appendix S7). The relative abundances of different fungal functional guilds also differed significantly between groups. Specifically, SPF had a significantly higher abundance of soil saprotroph and wood saprotroph compared to other groups (p < 0.05). However, SA and SK had significantly higher abundances of plant pathogen (p< 0.05) compared to that in SPF (Figure 5c).
The abundance of functional genes associated with nitrogen cycling, including nifB , nifH , nifU , and nirZ , in each 16S library was analyzed (Appendix Figure S6, Appendix S8). The relative abundances of these four genes of the SPF treatment showed no significant difference compared to other treatments, indicating that the nitrification metabolism in soil was not affected by pesticides, fertilizers, and/or excipients addition. We also found that the SF group with organic fertilizer addition, it significantly improved the abundance of soil beneficial bacteria Bacillus andPaenibacillus compared to the SA and SK groups without fertilizer addition (p < 0.05) (Figure 6). Brevibacilluswas also highly enriched in the SF group compared with that in the SA, SK, and SP groups (p < 0.01). The abundances of these three bacterial genera in SF also showed higher than other groups except for the SPF, which demonstrated that the SPF group with the organic fertilizer addition would be low harm to the soil environment when applied to the sugarcane field.