The objective of this study is to research the effect of broccoli residues (BR) on enzyme activity of cotton rhizosphere soil and relationships between enzyme activity and carbon source utilization capacity, and to provide a theoretical basis for revealing the ecological mechanism of BR to prevent Verticillium wilt disease and promote the growth of cotton.

In this study, soil treated with BR and soil treated without BR (CK) were set, and the activities of peroxidase, neutral phosphatase, arylsulfatase, chitinase, urease, β-glucosidase and N-acetyl-β-D-glucosaminidase in the rhizosphere soil of different treatments were determined, respectively. Shannon-Wiener index, Simpson index, Pielou index, McIntosh index, and richness index of different treatments were studied by Biolog-ECO technology. Principal component analysis (PCA) was used to compare the characteristics of microbial metabolic activity in rhizosphere soil under different treatments, and redundancy analysis (RDA) was used to analyze the correlations between soil enzyme activity and the utilization capacity of different types of carbon sources.

Compared with the CK, the plant height, branch and boll numbers treated with BR significantly increased by 12.73%, 16.95%, and 10.36%, respectively. The disease index of cotton Verticillium wilt treated with BR decreased by 64.19%. BR treatment significantly increased pH, nitrate nitrogen (NO₃^--N) and organic matter (OM) contents in rhizosphere soil. Meanwhile, there was no significant difference in the functional diversity index of rhizosphere microorganisms between treatments. However, BR changed the metabolic activities of rhizosphere microorganisms. BR treatment significantly increased the activity of enzymes related to cycles of different nutrient elements. In the aspect of carbon cycle, the activity of peroxidase and β-glucosidase was 2.70 and 1.95 times that of the control, respectively. In the nitrogen cycle, the activity of urease, chitinase and N-acetyl-β-D-glucosidase was 1.42, 1.59 and 1.52 times that of the control, respectively. In the phosphorus cycle, the activity of neutral phosphatase was 1.33 times that of the control. In the sulfur cycle, the activity of arylsulfatase was 1.22 times that of the control. RDA showed that soil enzyme activities under the treatment of BR were positively correlated with the utilization capacity of L-phenylalanine, L-threonine, glycogen, 2-hydroxybenzoic acid, itaconic acid, and D-malic acid, respectively.

BR changed the carbon metabolic activity of rhizosphere soil microorganisms, significantly increased the activity of soil nutrient cycling-related enzymes, pH, nitrate nitrogen, and organic matter contents in rhizosphere soil. Meanwhile, different degrees of correlations were found between soil enzyme activity and carbon metabolism characteristics.

Root exudates are signal mediators for the interaction between plants and soil microorganisms, which have important regulatory functions for plant disease occurrence and plant growth. The objective of this study is to clarify the microecological mechanism of L-proline in root exudates of cotton against the occurrence of Verticillium wilt, reveal the relationship of L-proline-mediated interaction between rhizosphere microorganisms and the occurrence of cotton Verticillium wilt, and to provide a new perspective for the construction of beneficial bacterial communities for the biological control of soil-borne diseases.

Through greenhouse pot experiments, different concentrations of L-proline (0, 50, 100, 200, and 400 mmol·L^-1) were set as experimental treatments, and real-time fluorescence quantitative PCR and metagenomic sequencing techniques were used to determine the DNA copy numbers of Verticillium dahliae and microbial community structure and function of soil samples treated with L-proline. The microbial community structures of rhizosphere soils under different concentrations of L-proline were compared using principal component analysis. Redundancy analysis was used to study the correlations between soil nutrient factors and microbial community structure, and Spearman correlation analysis was focused on the relationships between microbial community structure and functional metabolic pathways.

Compared with the blank control, the occurrence of cotton Verticillium wilt could not reduce under low concentration (50 mmol·L^-1) L-proline treatment, while the disease indexes under high-concentration (100, 200, and 400 mmol·L^-1) L-proline treatments were decreased by 22.51%, 60.23%, and 64.23%, respectively. The results of qPCR showed that L-proline treatment did not significantly decrease the copy numbers of V. dahliae in soil. The metagenomic sequencing analysis showed that the Shannon index of bacterial diversity significantly increased after L-proline treatment, while the Shannon index of fungal diversity showed a downward trend. At the genus level, the relative abundances of Nocardioides, Lysobacter, Arthrobacter, Phycicoccus, Pseudomonas, and Mucor were increased after treatment with L-proline. Linear discriminant analysis (LDA) showed that the enrichment of microbial KEGG pathways in rhizosphere soil changed after exogenous addition of L-proline, except for L-proline at the concentration of 100 mmol·L^-1. Redundancy analysis showed that bacterial community composition was significantly influenced by pH, conductivity, nitrate nitrogen, ammonium nitrogen, and organic matter (OM), while fungal community composition was significantly correlated with ammonium nitrogen. Spearman correlation analysis showed that the bacterial KEGG pathways were negatively correlated with pH, OM and ammonium nitrogen, while positively correlated with conductivity and nitrate nitrogen. Most of the KEGG pathways of fungi had poor correlation with soil nutrients.

The exogenous addition of an appropriate amount of L-proline can influence the occurrence of cotton Verticillium wilt through altering the structure and function of soil bacterial communities, increasing the relative abundance of beneficial microorganisms, but it cannot change the DNA copy numbers of pathogen. Meanwhile, the composition and function of bacterial community are related to soil nutrients.