Soil physicochemical properties at different growth stages
The physicochemical properties of all soil samples collected at 20, 40, 60, 80, and 100 days after the crops were planted at five different stages were measured in succession (Fig. 1). The soil pH of the rhizosphere soil samples from the monoculture soybean all decreased, while that of the corn samples increased; intercropped soybean and corn rhizosphere soil samples all decreased and then increased and showed the lowest value at the S3 stage (Fig. 1). The SOM contents in the monoculture and intercropped soybean rhizosphere soil samples all decreased and then increased, and the SOM content of the intercropped corn gradually increased (Fig. 1). The AP, AN, and AK contents in the intercropped soybean rhizosphere soil samples all decreased from the S1 to S3 stage and then rebounded to normal levels at the S3 stage. The AP and AK contents of the monoculture soybean showed no significant changes during the stages, and the AN contents all increased and then decreased; the AN and AK contents of the monoculture corn all decreased and then increased. The AP of the intercropping soybean increased and then rebounded to the normal level at the S3 stage (Fig. 1). The content of ACd in monoculture soybean rhizosphere soil samples all decreased and then increased, but the intercropped soybean soil samples showed the opposite trend. The dynamic trends of the physicochemical properties of the corn rhizosphere soil were opposite to those of the soybean rhizosphere soil (Fig. 1).
Bacterial Community Diversity And Composition At Different Growth Stages
After a series of steps, a total of 3,881,770 high-quality sequences were obtained from Illumina MiSeq sequencing of 80 rhizosphere soil samples. The observed richness (OTU numbers) and Shannon and inverse Simpson indices of monoculture and intercropped soybean rhizosphere soil samples all decreased and then increased, while monoculture and intercropped corn rhizosphere soil samples showed no significant changes at their different stages (Fig. 2). The Chao1 estimated richness of the monoculture and intercropped corn and soybean rhizosphere soil samples gradually increased at the five stages (Fig. 2). In addition, Student’s t-test results between the two groups showed significant differences in the α diversity indices between monoculture corn and soybean at the S2, S3 and S4 stages as well as intercropped corn and soybean at the S1, S2 and S4 stages (Table S2). There were also significant differences in the α diversity indices between monoculture and intercropped soybean at the S4 stage and monoculture and intercropped corn at the S2 stage (Table S2).
The principal coordinate analysis (PCoA) (Fig. 3) and PERMANOVA results (Table S3) showed that the monoculture soybean rhizosphere bacterial community structure was significantly different from S1 to S4, while that of the intercropped soybean was significantly different at the S1, S2, S3(S4) and S5 stages. The monoculture and intercropped corn rhizosphere bacterial community structure was significantly different among all five stages (P < 0.05). There was a significant difference in monoculture corn and soybean rhizosphere bacterial community structures among the five stages and in those of intercropped soybean and corn at the S1(S2), S3, S4, and S5 stages. The monoculture and intercropping soybean rhizosphere bacterial community structure was significantly different at the S1, S2 and S4 stages, and the monoculture and intercropped corn rhizosphere bacterial community structure was significantly different at the S2 and S3 stages.
Changes in bacterial taxa in rhizosphere soils at five different stages
The soil bacterial communities were significantly altered during the five different growth stages. The phyla Actinobacteria, Proteobacteria, Chloroflexi, Firmicutes, Acidobacteria, and Bacteroidetes accounted for 88.02 and 96.38% of those communities in soil and plant samples, respectively (Fig. 4). The relative abundance of the top 25 genera with significant difference in the soil is shown in Table S4. The relative abundance of Sphingomonas and Nocardioides in intercropped soybean in the S1 stage was significantly higher than that of monoculture soybean, and there were no significant differences in the abundance of other species. The relative abundance of Chryseobacterium, and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium in intercropped soybean in the S2 stage was significantly higher than that in monoculture soybean, and the relative abundances of Bradyrhizobium, Intrasporangium, Enterobacter, Acinetobacter, Microbacterium, Uncultured bacterium, Mycobacterium, and Ktedonobacter were significantly lower than those of monoculture soybean. The relative abundance of Bacillus, Fictibacillus and Oryzihumus in intercropped corn was significantly higher than that of monoculture corn. Metagenome, uncultured Acidobacteria bacterium, Burkholderia-Caballeronia-Paraburkholderia, and Bryobacter were significantly lower in abundance in intercropped than in monoculture corn, while there was no significant difference in other genera. In the S3 period, the relative abundance of Bacillus and Mesorhizobium in intercropped soybean was significantly higher than that of monoculture soybean. Enterobacter and Sphingobacterium showed a significantly lower relative abundance in intercropped than in monoculture soybean. The relative abundance of Streptomyces in intercropped corn was significantly lower than that in monoculture corn, and there was no significant difference in other genera. The relative abundance of Bradyrhizobium in the intercropped soybean samples was significantly higher than that in the monoculture soybean samples in S4. The relative abundances of Intrasporangium, Metagenome, Acinetobacter and Oryzihumus were significantly lower in intercropped soybean than in monoculture soybean, and the relative abundance of Streptomyces was significantly lower than that in monoculture corn, with no other significant differences. During the S5 period, the relative abundance of Enterobacter microbes in intercropped soybean was significantly lower than that in monoculture soybean, while that of Metagenome and Luedemannella was significantly lower than that of monoculture corn, and there were no other significant differences.
The Relationship Between Physicochemical Properties And The Bacterial Community
The mantel test was performed to examine the correlation between physicochemical properties and bacterial community composition. The results showed that pH and SOM were significantly correlated with the rhizosphere soil bacterial communities (P < 0.05, Table 1). The SOM had the highest correlation with the rhizosphere soil bacterial community (Bray-Curtis distance, r = 0.2756, P = 0.001), with no significant correlation found between AP, AN, AK, ACd and the rhizosphere soil microbial community. To determine the relative contribution of environmental variables to the bacterial community, canonical correspondence analysis (CCA) and CCA-based variation partitioning analysis (VPA) were further performed. CCA-based VPA showed that pH, SOM, ACd, and available (P, K, N) explained 2.82%, 3.43%, 1.34% and 5.43% of the variation in the rhizosphere soil bacterial community, respectively. Their interaction could explain 3.02% of the variation, leaving 83.96% of the variation unexplained (Fig. 5).
Table 1
Mantel analysis of the relationship between the microbial community structure and soil properties. P-values were calculated using the distribution of the Mantel test statistics estimated from 9999 permutations. *:P < 0.05, **:P < 0.01, ***:P < 0.001.
Soil properties | Bray–Curtis | Jaccard |
r | p | r | p |
pH | 0.1344 | 0.037* | 0.1456 | 0.024* |
SOM | 0.2756 | 0.001*** | 0.293 | 0.001*** |
AP | -0.0505 | 0.72 | -0.0271 | 0.585 |
AN | -0.0366 | 0.699 | -0.0237 | 0.619 |
AK | -0.0783 | 0.879 | -0.0522 | 0.766 |
ACd | -0.0319 | 0.573 | -0.0431 | 0.695 |