Additional file 1 of Redundancy in microbiota-mediated suppression of the soybean cyst nematode

Supplementary Material 1: Table S1. Physical and chemical properties of SCN suppressive and conducive field soils used in this study. Table S2. Primers used to quantify the transcript levels of GH18 genes of Chitinophaga isolate CN7 by qRT-PCR. Table S3. Primers used to amplify three GH18 genes of Chitinophaga isolate CN7 for protein production in Escherichia coli. Table S4. Primers used to quantify the transcript levels of selected defense-related genes in soybean via qRT-PCR. Fig. S1. SCN egg densities and soybean growth in the suppressive (S) and conducive (C) soils collected from soybean fields in Baicheng and Flaerji. A. Egg densities in the S and C soils. B. Soybean shoot weight after 56 days of growth using the S and C soils in a growth chamber. C. Cyst densities and soybean shoot weight after 56 days of growth in the following soils inoculated with 1,500 eggs per 100g of dried soil: S and C soils, transferred soil (CS; created by mixing 10% S soil with 90% C soil), heat (80°C)-treated S soil (S80), and S soil fumigated with formalin (SF). Different letters above the bars denote statistically significant differences according to LSD test (p < 0.05). Fig. S2. SCN suppression by different field soils and soil treatments. Greenhouse trials were performed to determine how various soils and soil treatments affect SCN egg densities and whether bacteria and fungi in the rhizosphere and SCN cyst contribute to suppressing SCN. The suppressive (S) and conducive (C) soils collected from Baicheng (A) and Fulaerji (B) were used. The egg densities were measured after 56 days growth of soybean with inoculation of 1,500 eggs/100g dried soil. The classical trial for suppressive soil included the treatments: suppressive (S) soil, conducive (C) soil, transferred soil (CS; created by mixing 10% S soil with 90% C soil), heat (80°C)-treated S soil (S80), and S soil fumigated with formalin (SF). To illustrate contributions of cysts (Cy) and soybean rhizosphere (Rh) microbiota in SCN suppressiveness, microbial suspensions were prepared from the cysts and rhizosphere in the S soil equal to 10% amount S soil in CS treatment and then mixed with 90% C soil. The rhizosphere microbial suspensions were prepared by collected rhizosphere (including roots) soil from soybean seedlings growing in S soil equal to 10% S soil in CS treatment for two weeks and ground in distilled water (CS-Rh). Cysts microbial suspensions were created by extracting cysts from native S soil equal to amount of 10% S soil in the CS treatment and ground in distilled water (CS-Cy). The microbial suspensions were also treated with antibiotics to kill fungi (pimafucin=200 μg/mL) and remain bacteria (CS-Rh-bacteria or CS-Cy-bacteria) or to kill bacteria (penicillin=100 U/mL+streptomycin=100 μg/mL) and remain fungi (CS-Rh-fungi or CS-Cy-fungi) or to kill all microbiota by 3 antibiotics (CS-Rh-antibiotics or CS-Cy-antibiotics). Different letters above the bars denote statistically significant differences according to LSD test (p < 0.05). Fig. S3. Within-sample diversity (α-diversity) of bacterial community inhabiting the cysts isolated from five soil treatments. A. Bacterial community cluster analysis based on Bray–Curtis dissimilarity showed clear separation between SCN cyst in S and CS soil treatments than C, S80 and SF (left in Baicheng and Flaerji). The horizontal bar within each box indicates median value. The tops and bottoms of boxes represent 75th and 25th quartiles, respectively. Linear regression relationship between the cyst bacteria diversity and egg densities. Regression line is in brown, and the shaded region represents the confidence interval (geom_smooth function, method = lm). B. Cluster analysis based on Bray–Curtis dissimilarity indicates clear separation between bacterial communities inhabiting the SCN cyst in S and CS soil treatments than C, S80, and SF. Bacterial OTUs with RA > 1‰ in at least one sample were included in the analysis. Fig. S4. Averaged relative abundance (RA) and linear regression relationship between the most dominant bacterial phyla and the egg density. Data from analysing the S and C field soils collected in (A) Baicheng and (B) Fulaeji were used for this analysis. The brown line and the shaded region on the right side indicate the regression line and the confidence interval (geom_smooth function, method = lm), respectively. Only the taxa with RA of > 1% in at least one sample were included in the analysis. Fig. S5. Average relative abundance (RA) of the most dominant bacterial families associated with the cysts formed in different soils. Chitinophagaceae was more abundant in the cysts formed in the S and CS soils than those formed in the C soils collected from two provinces (A=Beicheng, B=Fulaerji). Only taxa with RA of > 1% in at least one sample were included. Fig. S6. Ternary plots and heatmaps demonstrating the OTUs significantly enriched in heat (S80)- and formalin (SF)-treated soils compared to S across two district geographical locations (Baicheng-BC, A and B; Fulaerji-FL, C and D). Dark golden circles mark cyst OTUs significantly enriched in S80 than S (S80 > S OTUs). Pink circles mark cyst OTUs significantly enriched in SF than S (SF > S OTUs). Green circles mark cyst OTUs simultaneously enriched in S80 and SF compared to S (S80+SF > S OTUs). Each circle represents one OTU. The size of each circle represents its relative abundance. The position of each circle is determined by the contribution of the indicated compartments to the total relative abundance. Only taxa with RA > 1‰ in at least one sample were included in the analysis. Fig. S7. Detection and quantification of Chitinophaga in the suppressive soil and cysts by qPCR. A. The gel image shows specific amplification of the targeted Chitinophaga DNA region using the primer pair hu3135254F (5′-CATTGAGAGGCATCTTTTG-3′) and hu1185451R (5′-CGGTGCTTATTCATCTGGTA-3′). M indicates DNA size markers (2000 bp). Lanes 1-6 show PCR amplification products in the absence of Chitinophaga genomic DNA. Lanes 7-12 show PCR amplification products in the presence of Chitinophaga genomic DNA. B. Estimation of the percentage of Chitinophaga among the bacterial cells in the suppressive soil and those associated with cysts. The primers 27F and 1492R, designed to amply the bacterial 16S rRNA genes, were used to quantify the total bacterial community size in each sample. (C) Chitinophaga RA % in rhizosphere, root and cyst of suppressive soils challenged with nematodes in a previous study by Hussain et al. [1]. Fig. S8. The circos plot displaying the genomic features of chromosomal level Chitinophaga isolate CN7 genome. COG functional gene distribution. The legend is shown on the right side. Fig. S9. The circos plot displaying the genomic features of chromosomal level Chitinophaga isolates C54 genome. COG functional gene distribution. The legend is shown on the right side. Fig. S10. Relative expression of chitinases encoded by three glycosyl hydrolase 18 (GH18) genes of Chitinophaga isolate CN7 challenged by SCN eggs and their heterogeneous expression using Escherichia coli BL21 and their purification. The PCR primers used for expression vector construction are listed in Table S3. A. Expression of GH18 genes after 0, 24 and 48 h by inoculation of 1 ml CN7 cell suspension (109 cfu/mL) to 100 SCN eggs. B. SDS-PAGE of cell lysates before and after IPTG induction. The bands corresponding to target proteins were marked in black boxes. Proteins in the whole cell lysate (A) and the supernatant (S) and precipitate (R) of the lysate after ultra-sonication and centrifugation were analyzed. C. SDS-PAGE of purified proteins. The target protein bands are marked in black box. M=molecular weight (kDa) markers; T=the cell lysate before passing through Ni-NTA; 1-2=elutants with 50 mM imidazole buffer; 3-7=elutants with 150 mM imidazole buffer; 8=elutant with 500 mM imidazole buffer.