Pre-soil fumigation with ammonium bicarbonate and lime modulates the rhizosphere microbiome to mitigate clubroot disease in Chinese cabbage

Abstract

Background: Plasmodiophora brassicae is an ever-increasing threat to cruciferous crop production worldwide.

Aims and methods: This study investigated the impact of pre-soil fumigation with ammonium bicarbonate (N) and lime (NB) to manage clubroot disease in Chinese cabbage through 16S rRNA gene amplification sequencing.

Results: We found that soil fumigation with N and NB suppressed disease incidence by reducing the soil acidity and population of P. brassicae in the rhizosphere. Minimum disease incidence and maximum relative control effect of about 74.68 and 66.28% were achieved in greenhouse and field experiments, respectively, under the combined application of ammonium bicarbonate and lime (LNB) as compared with N, NB, and control (GZ). Microbial diversity analysis through Miseq sequencing proved that pre-soil fumigation with N, NB, and LNB clearly manipulated rhizosphere microbial community composition and changed the diversity and structure of rhizosphere microbes compared with GZ. Bacterial phyla such as Proteobacteria, Bacteriodetes, and Acidobacteria and fungal phyla including Olpidiomycota and Ascomycota were most dominant in the rhizosphere of Chinese cabbage plants. Soil fumigation with N and NB significantly reduced the abundance of clubroot pathogen at genus (Plasmodiophora) level compared with GZ, while decreased further under combined application LNB. Microbial co-occurrence network analysis showed a highly connected and complex network and less competition for resources among microbes under combined application LNB.

Conclusion: We conclude that for environmentally friendly and sustainable agriculture, soil fumigation with combined ammonium bicarbonate and lime plays a crucial role in mitigating Chinese cabbage clubroot disease by alleviating soil pH, reducing pathogen population, and manipulating the rhizosphere microbiome.

Keywords: Plasmodiophora brassicae; biological control; disease suppression; rhizosphere microbiome; soil pH.

Figure 1

Impact of soil fumigation on clubroot disease of Chinese cabbage. Disease incidence (A), disease index (B), and control effect (C) under greenhouse conditions. Disease incidence (D), disease index (E), and control effect (F) under field conditions. Different small letters on the error bar show the significant difference among treatments according to the least significant difference (LSD) test at p < 0.05. Soil fumigation with lime (NB), soil fumigation with ammonium bicarbonate (N), combined soil fumigation with ammonium bicarbonate and lime (LNB), and non-fumigated soil (GZ).

Figure 2

Effect of soil fumigation on soil pH. Soil pH after 15 days of fumigation (A) and after harvesting the crop (B) in the greenhouse experiment. Soil pH after 15 days of fumigation (C) and after harvesting the crop (D) in the field experiment. Significance difference among treatments (as described in Figure 1) is shown by different small letters on the error bar according to the least significant difference (LSD) test at p < 0.05.

Figure 3

Population dynamic of clubroot pathogen Plasmodiophora brassicae in rhizosphere soil of Chinese cabbage plants under different treatments. P. brassicae copies per gram of soil under greenhouse (A) and field (B) conditions. According to the least significance test (LSD; p < 0.05), the different lowercase letters on the error bars showed a significant difference among treatments (as described in Figure 1).

Figure 4

Principal coordinate analysis (PCoA) based on the Bray–Curtis dissimilarity metric showing the beta diversity metrics of rhizosphere bacterial and fungal communities under different treatments. Weighted and Unweighted UniFrac PCoA analysis of rhizosphere bacterial (A,B) and fungal (C,D) community structure under different treatments (as described in Figure 1).

Figure 5

Operational taxonomic units (OTUs) analysis of bacterial and fungal communities under different treatments. Bar plots displaying the total numbers of bacterial (A) and fungal (B) OTUs under different treatments (as described in Figure 1). Venn diagram showing the unique and shared bacterial (C) and fungal (D) OTUs among the treatments (as described in Figure 1).

Figure 6

Relative abundance (RA) analysis of the most abundant bacterial and fungal phyla in the rhizosphere soil of Chinese cabbage. RA of top 15 bacterial (A) and fungal (B) phylum under different treatments. The bar plot shows the significant difference in RA of the differentially abundant bacterial (C) and fungal (D) phyla under different treatments. The significant difference in the RA of specific phylum among treatments (as described in Figure 1) is shown by different lowercase letters on the error bars according to the least significance difference test (LSD) p < 0.05.

Figure 7

Relative abundance (RA) bar plots of most abundant genera under different treatments. RA bar plots of bacterial (A) and fungal (B) genera level. Significant difference in the RA of specific genera among the treatments (as described in Figure 1) is shown by different small letters on the error bar according to the least significant difference (LSD) test at p < 0.05.

Figure 8

Relative abundance heatmaps for the top 20 bacterial (A) and fungal (B) OTUs in the rhizosphere of Chinese cabbage plants under different treatments (as described in Figure 1).

Figure 9

Co-occurrence network of bacterial and fungal communities under different treatments (as described in Figure 1) at the OTU level. Only p < 0.05 and corrections coefficients >0.6 (Spearman correlation) were constructed in the network. Red circles represent the bacterial OTUs, and green boxes represent the fungal OTUs. Red and blue edges indicate positive and negative correlations, respectively. Collinear network analysis of relationships between samples and species. Collinear network analysis of relationships between samples with bacteria phyla (A). Collinear network analysis of relationships between samples with fungal phyla (B).

Figure 10

The concluding sketch demonstrates the impact of soil fumigation on the occurrence of clubroot disease and the rhizosphere microbiome of Chinese cabbage plants. Diseased plants under no soil fumigation display high disease incidence, high pathogen population, low microbes, and low soil pH. Healthy plants under lime and ammonium bicarbonate soil fumigation represent high soil pH, high microbes, low pathogen population, and low disease incidence.