Cyanobacterium Nostoc species mitigate soybean cyst nematode infection on soybean by shaping rhizosphere microbiota

Abstract

Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is the most devastating and yield-limiting pathogen that threatens soybean production globally. Sustainable SCN disease management strategies are needed. In this study, a cyanobacterial strain was isolated from SCN-infected soybean soil and identified as Nostoc punctiforme using the cyanobacterial 16S rRNA gene sequence. When susceptible soybean plants were grown in the SCN-inoculated soil, N. punctiforme inoculants significantly reduced the total number of SCN eggs and second-stage juveniles (J2s), compared to the control with SCN inoculation only. Further microbial analysis showed that N. punctiforme inoculants changed the bacterial and fungal communities in the soybean rhizospheres and significantly increased the relative abundance of several bacterial and fungal species with potential nematicidal activities, suggesting the changes of soybean rhizosphere microbiota may partially contribute to the activity of N. punctiforme inoculants against SCN. However, N. punctiforme inoculants did not directly induce soybean defense reactions against SCN. Thus, N. punctiforme may be a potential microbial source against SCN invasion in soybean.

Keywords: cyanobacteria; microbiota; nematicidal activity; plant defense system; soybean cyst nematode.

Figure 1

Effect of Nostoc punctiforme inoculants on soybean cyst nematode. CK: control without SCN (HG type 7) and N. punctiforme inoculation, SCN: SCN inoculation, Low Conc: SCN and low concertation N. punctiforme inoculation, High Conc: SCN and high concertation N. punctiforme inoculation. Different letters indicate a statistically significant difference between treatments within an experiment as determined by Kruskal-Wallis's test (p ≤ 0.05, n = 4).

Figure 2

Nonmetric multidimensional scaling (NMDS) plots of Bray-Curtis distances among soybean rhizosphere microbiota following SCN and N. punctiforme inoculation. Samples are colored by SCN and N. punctiforme inoculation. (A) bacterial community, (B). fungal community. SCN: SCN inoculation, Low Conc: SCN and low concertation N. punctiforme inoculation, High Conc: SCN and high concertation N. punctiforme inoculation. The different groups are highlighted by ellipses showing a 95% confidence range for the variation within each group.

Figure 3

Differentially abundant bacterial ZOTUs identified in the soybean rhizosphere by SCN and N. punctiforme inoculations (FDR adjusted p < 0.1, Wald's test). (A) Bacterial ZOTUs in the SCN and low concertation N. punctiforme inoculation, (B) bacterial ZOTUs in the SCN and high concertation N. punctiforme inoculation. SCN: SCN inoculation, Low Conc: SCN and low concertation N. punctiforme inoculation, High Conc: SCN and high concertation N. punctiforme inoculation. Values on the x-axis presented the DESeq2-estimated log2-fold difference. The red vertical line represents a zero-fold change, where ZOTUs to the right of the line (positive values) are increased in relative abundance in N. punctiforme inoculation, and those to the left of the line (negative values) are more abundant in no N. punctiforme inoculation. Dots indicate ZOTUs, where the size of the dot is scaled by its mean abundance among all samples (base mean ≥ 20) and its color represents the phylum to which that ZOTUs belongs.

Figure 4

Differentially abundant fungal ZOTUs identified in the soybean rhizosphere by SCN and N. punctiforme inoculations (FDR adjusted p < 0.1, Wald's test). (A) Fungal ZOTUs in the SCN and low concertation N. punctiforme inoculation, (B) fungal ZOTUs in the SCN and high concertation N. punctiforme inoculation. SCN: SCN inoculation, Low Conc: SCN and low concertation N. punctiforme inoculation, High Conc: SCN and high concertation N. punctiforme inoculation. Values on the x-axis presented the DESeq2-estimated log2-fold difference. The red vertical line represents a zero-fold change, where ZOTUs to the right of the line (positive values) are increased in relative abundance in N. punctiforme inoculation, and those to the left of the line (negative values) are more abundant in no N. punctiforme inoculation. Dots indicate ZOTUs, where the size of the dot is scaled by its mean abundance among all samples (base mean ≥ 20) and its color represents the phylum to which that ZOTUs belongs.

Figure 5

RT-qPCR analysis of the expression of pathogenesis-related genes. (A) Relative expression of PR1, (B) Relative expression of PR2, (C) Relative expression of PR3, (D) Relative expression of PR5, (E) Relative expression of PR10. Gene expression levels were analyzed from four treatments: CK: control without SCN and N. punctiforme inoculations, SCN: SCN inoculation, Low Conc: SCN and low concertation N. punctiforme inoculations, High Conc: SCN and high concertation N. punctiforme inoculations. Data are normalized by the ubiquitin reference gene and relative transcript levels in comparison with those of CK. Error bars represent standard error of the mean values of two technical replicates from four biological replicates. Differences in expression levels were tested by analysis of variance (ANOVA) and Tukey test. For each gene, bars with different letters indicate statistically significant differences between treatments.

Figure 6

Effect of N. punctiforme co-existing microbial inoculants on soybean cyst nematode. SCN: SCN inoculation, Stenotrophomonas: SCN and Stenotrophomonas maltophilia inoculation, Paenibacillus: SCN and Paenibacillus lactis inoculation, Nocardioides: SCN and Nocardioides zeae inoculation, Pseudomonas: SCN and Pseudomonas sp. inoculation, Unclassified fungus: SCN and unclassified fungus inoculation (Kruskal-Wallis's test, p = 0.72, n = 4).