An Endophytic Strain of Bacillus amyloliquefaciens Suppresses Fusarium oxysporum Infection of Chinese Wolfberry by Altering Its Rhizosphere Bacterial Community

Abstract

Root rot disease is a serious infection leading to production loss of Chinese wolfberry (Lycium barbarum). This study tested the potential for two bacterial biological control agents, Bacillus amyloliquefaciens HSB1 and FZB42, against five fungal pathogens that frequently cause root rot in Chinese wolfberry. Both HSB1 and FZB42 were found to inhibit fungal mycelial growth, in vitro and in planta, as well as to promote the growth of wolfberry seedlings. In fact, a biocontrol experiment showed efficiency of 100% with at least one treatment involving each biocontrol strain against Fusarium oxysporum. Metagenomic sequencing was used to assess bacterial community shifts in the wolfberry rhizosphere upon introduction of each biocontrol strain. Results showed that HSB1 and FZB42 differentially altered the abundances of different taxa present and positively influenced various functions of inherent wolfberry rhizosphere bacteria. This study highlights the application of biocontrol method in the suppression of fungal pathogens that cause root rot disease in wolfberry, which is useful for agricultural extension agents and commercial growers.

Keywords: Bacillus; F. oxysporum; biocontrol; rhizosphere bacterial community; root rot; wolfberry.

FIGURE 1

Phylogenetic tree based on 16S rRNA gene showing relationships between Bacillus strain HSB1 and other Bacillus strains/species derived from NCBI accessions. The tree was inferred using the neighbor-joining method and MEGA 5.5 software with 1000 bootstrap replicates (bootstrap values are shown next to nodes). The strain HSB1 clustered with B. amyloliquefaciens, B. methylotrophicus, and B. velezensis strains, (a). B. mojavensis strains were used as out-group (b).

FIGURE 2

Plate assay showing antagonistic activity of both B. amyloliquefaciens FZB42 and HSB1 against each of five fungal pathogens of Chinese wolfberry: F. solani, A. alternata, F. tricinctum, F. oxysporum and F. chlamydosporum (A–E). Panels (F–J) are the respective controls.

FIGURE 3

Bar graphs showing changes in stem length (A), and root length (B) of wolfberry seedlings whose soil was treated with Bacillus amyloliquefaciens strains (FZB42 or HSB1) compared to a water control (CK). Measurement time points are based on growth at 60, 68, and 76 day. Error bars represent standard deviation of three replicates.

FIGURE 4

Bar graphs showing changes in shoot weight (A,B), and root weight (C,D) of wolfberry seedlings whose soil was treated with Bacillus amyloliquefaciens strains (FZB42 or HSB1) compared to a water control (CK). Measurement time points are based on days after inoculation growth at 7, 14, and 21 days after inoculation. Error bars represent standard deviation of three replicates. Letters above each bar indicate significant differences from the control (CK) (p < 0.05).

FIGURE 5

Bar charts showing disease incidence (A–C), blue bars) and disease severity (D–F), orange bars) for all treatments recorded at 15, 21, and 25 days time points. Error bars represent standard error of three replicateds. Letters above each bar indicate significant differences in disease incidence at 25 days between F. oxysporum, HSB1 + F. oxysporum, and F. oxysporum + FZB42, and treatments (p < 0.05).

FIGURE 6

Rarefaction curves (A) showing the relationship between sequence number per sample and observed OTUs for different treatment replicates. The length of the curve reflects sequencing depth (i.e., a longer curve indicates a greater depth), while the smoothness reflects the effect of sequencing depth on sample diversity. Principal component analysis (B) based on the distance matrix calculated using the Bray-Curtis algorithm for soil samples collected from different treatments: CK, control; F, F. oxysporum alone; FH, F. oxysporum + HSB1; FZ, F. oxysporum + FZB42; H, HSB1 alone; HF, HSB1 + F. oxysporum; Z, FZB42 alone; ZF, FZB42 + F. oxysporum.

FIGURE 7

Stacked bar charts showing the relative abundances (in different colors) of the top 10 classified bacterial phyla (A) and genera (B) detected in soil samples subjected to different treatments: CK, control; F, F. oxysporum alone; FH, F. oxysporum + HSB1; FZ, F. oxysporum + FZB42; H, HSB1 alone; HF, HSB1 + F. oxysporum; Z, FZB42 alone; ZF, FZB42 + F. oxysporum. Relative abundance was based on the proportional frequencies of DNA sequences classified at the phylum and genus levels. Length of a color bar correlates with amount of abundance. Data were averaged from three replicates of each treatment.

FIGURE 8

Metagenome comparisons, as predicted by PICRUSt, showing significant differences in the functionality of microbial genes detected in soil samples collected from selected treatments: CK, control; F, F. oxysporum alone, H, HSB1 alone, and FZ, F. oxysporum + FZB42. (A): significant comparisons between CK and H treatments, (B): significant comparisons between F and H treatments, and (C): significant comparisons between H FZ treatments.