Effect of Cd on physiological properties of oilseed rapes and Cd accumulation
Compared to the control (CK), Cd treatment suppressed plant growth (Fig. 1), specifically plant height, fresh weight, and total leaf area were significantly reduced with increasing Cd concentration (P<0.05). At CK and lower Cd concentration (10 mg/kg), the biomass of B. napus was significantly higher than B. juncea, but at higher Cd concentration (30 mg/kg) those tendencies were reversed. Pearson correlation analysis showed that Cd content in tissues was negatively correlated with plant height, weight, and leaf area in B. napus and B. juncea (P<0.01) (Additional file 1: Table S1 and Table S2).
Chlorophyll was significantly decreased under higher Cd concentration, with B. juncea being significantly less affected than B. napus (P<0.05) (Fig. 1D). Soluble sugar content was markedly decreased under Cd stress in both of B. napus and B. juncea, while the content of soluble protein was markedly increased under Cd treatment when compared with CK (P<0.05) (Fig. 1E , F). SOD and POD activity values showed similar trends, decreasing at first and then increasing. The SOD activity at 30 mg/kg was significantly higher than in other treatments (P<0.05). However, CAT activity was notably inhibited at the higher Cd concentration (P<0.05). SOD and POD activity were higher in B. napus than B. juncea, while CAT activity values were the reverse (Fig. 1G, H, I). Pearson correlation test showed that plant Cd content was negatively related with chlorophyll, soluble sugar, and CAT (P < 0.01) and positively related with soluble protein and SOD (P < 0.05) in B. napus and B. juncea (Additional file 1: Table S1 and Table S2).
The concentration of Cd in oilseed rape leaves and roots were significantly higher with increasing Cd levels, and Cd content in B. napus tissues was significantly higher than B. juncea (P<0.05) (Additional file 2: Figure S1). TF in the two oilseed rapes species significantly decreased with increasing Cd and was higher in B. napus than B. juncea (P<0.05) (Additional file 1: Table S3). BAF in leaves and roots of both B. napus and B. juncea was bigger than 1 and higher in 30 mg/kg compared to 10 mg/kg, especially in roots (Additional file 1: Table S3).
Effect of Cd on physicochemical properties of soils
In B. napus, the pH, TN, TP, and NO3_N were significantly decreased under the higher Cd concentration (P<0.05) and TOC first increased and then decreased in both rhizosphere and bulk soils (Fig. 2), meanwhile TOC, TN, NH3_N, and AP were higher, and pH lower, in rhizosphere than bulk soil. pH and TN were markedly reduced in the 30 mg/kg Cd treatment (P<0.05) and NH3_N was first increased and then decreased in both rhizosphere and bulk soils, while AK was significantly increased in bulk soil (P<0.05) in B. juncea under the higher Cd concentration (Fig. 2). Furthermore, TOC, TN, TP, NH3_N, NO3_N, and AK were higher, and pH was lower, in rhizosphere than bulk soil. Compared with B. napus, most soil nutrients were higher in B. juncea samples under the higher Cd treatment.
Pearson correlation analysis showed that for B. napus, the Cd content was negatively correlated with pH, TN, TP, and NO3_N content in both rhizosphere and bulk soils and with TOC of the rhizosphere (Additional file 1: Table S4 and Table S6). Cd was negatively correlated with pH, TN, and NH3_N content in rhizosphere and bulk soils and negatively correlated with AP of the rhizosphere, but positively correlated with AK of bulk soil in B. juncea (P < 0.05) (Additional file 1: Table S5 and Table S7).
Effect of Cd on bacterial numbers in soils
The results revealed that total bacterial numbers slightly increased with increasing Cd levels (Additional file 2: Fig. S2) and the Pearson analysis showed that Cd was positively correlation with bacterial numbers in bulk soil of B. napus (Additional file 1: Table S8). Meanwhile, no significant difference was observed between B. napus and B. juncea (P > 0.05).
Effect of Cd on the α-diversity of bacterial community
After removing low quality reads and chimaeras in 108 plant samples and 72 soil samples, a total of 13,352,813 high-quality 16s rRNA gene reads were obtained, which were clustered into 14359 phylotypes (OTUs) by grouping at a 97% identity threshold. The sequencing depths of all samples were appropriate for downstream analyses (Additional file 2: Fig. S3).
In the plant samples, Cd mainly affected root endophytic community of B. napus and phyllosphere community of B. juncea. Shannon index and richness of B. napus’s root endophytes and the richness and Chao1 of B. juncea’s phyllosphere decreased significantly at the higher Cd concentration (P<0.05) (Additional file 2: Fig. S4). Pearson correlation analysis demonstrated that plant physiological factors mainly correlated with α-diversity indexes of root endophytes in B. napus (Additional file 1: Table S10) and that the majority of plant physiological properties were cardinally correlated with OTU numbers (richness and Chao1) of phyllosphere in B. juncea (Additional file 1: Table S11). pH and TOC were significantly positive correlated with and Cd was significantly negative correlated with the α-diversity of root endophytic bacterial communities in B. napus (Additional file 1: Table S10).
However, high level of Cd significantly depressed the α-diversities in the soil bacterial communities. Inverse Simpson index and richness of rhizosphere and inverse Simpson indexes of bulk soil were markedly reduced in the 30 mg/kg Cd treatment in B. napus (P<0.05) (Fig. 3). Shannon and inverse Simpson indexes of rhizosphere and Shannon, inverse Simpson, richness and Chao1 of bulk soils in B. juncea were significantly decreased under the higher Cd treatment (P<0.05) (Fig. 3). Most α-diversity indexes between the two species of oilseed rapes had no significant differences (Fig. 3 and Additional file 2: Fig. S4).
Pearson tests showed that plant’s physiological factors mainly influenced α-diversity indexes of the rhizosphere in B. napus and bacterial diversity (Shannon and inverse Simpson indexes) of the rhizosphere in B. juncea (Additional file 1: Table S12 and Table S13). pH, TOC, TN, TP, and NO3_N were positively correlated with the α-diversity in rhizosphere of B. napus. Meanwhile, TN and NO3_N were positively correlated with bacterial diversity (Shannon and inverse Simpson indexes) of bulk soil in B. napus samples (Additional file 1: Table S12). In B. juncea, pH was positively, and AK negatively, correlated with α-diversity in bulk soil (Additional file 1:Table S13). However, Cd concentration showed a significant negative correlation with α-diversity of soil bacteria communities for both species of oilseed rapes (P<0.05) (Additional file 1: Table S12 and Table S13).
Effect of Cd on bacterial community composition and structure
Cd could affect the composition of bacterial communities in soil-plant ecosystem, particularly under higher levels of Cd stress (Additional file 1: Table S14, Table S15, Table S16, Table S17 and Table S18). Under 30 mg/kg Cd treatment, the bacterial relative abundance on the phylum level of Gemmatimonadetes, and Chloroflexi were significantly decreased in the B. juncea phyllosphere (P<0.05). Actinobacteria was significantly decreased in B. juncea’s root endophytic community and the B. napus rhizosphere (P<0.05). Gemmatimonadetes and Verrucomicrobia were significantly reduced in both rhizosphere and bulk bacterial community (P<0.05), but Proteobacteria and Bacteroidetes were significantly increased in the B. napus rhizosphere and Firmicutes was significantly increased in bulk soil of both of two rapeseed species.
At the genus level (Fig. 4), the relative abundance of some genera was altered under Cd treatment. In the B. napus phyllosphere samples, the relative abundances of Massililia sp., Rhodanobacter sp., and Rickettsia sp. were increased, and Buchera sp., Achromobacter sp., and Acinetobacter sp. were decreased under Cd treatment. While in B. juncea phyllosphere samples, Lysobacter sp., Stenotrophomonas sp., and Gibbsiella sp. were increased, while Gaiell sp., Telluria sp., and Herbaspirillum sp. were decreased under Cd treatment. In leaf endophyte samples, Brochothrix sp. and Acinetobacter sp. were increased in B. napus but decreased in B. juncea under Cd treatment. In root endophyte samples, Chryseobacterium sp. and Pantoea sp. were increased and Caulobacter Ideonella sp. and Herbaspirillum sp. were decreased in B. napus. Sphingomonas sp., Ralstonia sp., and Methylobacterium sp. were increased, and Rhizobium sp., Rhodanobacter sp., and Duganella sp. were decreased in B. juncea under Cd treatment.
In the rhizosphere, Niastella sp., Methylotenera sp., and Lystobacter sp. were increased and Arthrobacter sp., Gemmatimanas sp., and Haliangium sp. were decreased in B. napus under 30mg/kg Cd treatment. Massilia sp., Ralstonia sp., and Streptomyces sp. were increased and GP2 sp., Terriglobus sp., and Candidatus Solibacter sp. were decreased in B. juncea under 30mg/kg Cd treatment. In bulk soil, Sphingomonas sp., Rhodanobacter sp., and Roseateles sp. were increased and Arthrobacter sp., Gemmatimonas sp., and Terriglobus sp. were decreased in B. napus. Streptomyces sp., Pseudomocardia sp., and Blastococcus sp. were increased and Haliangium sp., Phenylobacterium ap., and Gemmatimonas sp. were decreased in B. juncea under Cd treatment.
The principal co-ordinates analysis (PCoA) (Fig. 5) and dissimilarity analysis (Additional file 1: Table S19 and Table S20) indicated that the bacterial community structures of both rhizosphere and bulk in both B. napus and B. juncea were significantly changed under higher Cd concentration compared to control (P < 0.05), but not significantly affected plant bacterial community structures.
Relationship between microbial community structure and environmental factors
The result of Mantel test showed that there are no significant association between most environment factors and phyllosphere or leaf endophyte bacterial communities (Additional file 1: Table S21 and Table S22). Biomass (height, weight, and leaf area), TOC and root Cd had significant association with root endophyte bacterial community in B. napus (Additional file 1: Table S21). Biomass, pH, NO3_N and Soil_Cd had significant association with rhizosphere soil and TN and Soil_Cd had significant association with bulk soil in B. napus (Table 1). Biomass, TN and Soil_Cd were significant correlated with rhizosphere soil bacterial communityin B. juncea and pH and Soil_Cd were significant correlated with bulk soils bacterial community in B. juncea (Additional file 1: Table S23).
The CCA model of root endophyte, rhizosphere and bulk soil bacterial community were significant (P<0.05, Additional file 2: Fig. S5A, Fig. S6A and Fig. 6A). The results of VPA indicated that biomass, pH, soil nutrients, and Root_Cd explained 14.3%, 5.4%, 37.4%, and 7.2% of variation in B. napus (Additional file 2: Fig. S5B) and 15.5%, 6.9%, 40.2%, and 4.2% of variation in B. juncea (Additional file 2: Fig. S5C) in root endophyte bacterial community, respectively.
CCA-based VPA indicated that biomass, pH, soil nutrients, and Cd concentration of rhizosphere soil bacterial community explained 13.8%, 4.7%, 32.6%, and 3.6% of variation in B. napus (Additional file 2: Fig. S6B), and 14.7%, 3.4%, 35.7%, and 5.3% of variation in B. juncea (Additional file 2: Fig. S6C), respectively. For bulk soils bacterial community, VPA indicated that pH, soil nutrients, and Cd explained 4.9%, 39.4%, and 4.4% variation in B. napus (Fig. 6B) and 5.1%, 42.3%, and 6.1% variation in B. juncea (Fig. 6C), respectively.