Plant Growth Promotion and Plant Disease Suppression Induced by Bacillus amyloliquefaciens Strain GD4a

Abstract

Botrytis cinerea, the causative agent of gray mold disease (GMD), invades plants to obtain nutrients and disseminates through airborne conidia in nature. Bacillus amyloliquefaciens strain GD4a, a beneficial bacterium isolated from switchgrass, shows great potential in managing GMD in plants. However, the precise mechanism by which GD4a confers benefits to plants remains elusive. In this study, an A. thaliana-B. cinerea-B. amyloliquefaciens multiple-scale interaction model was used to explore how beneficial bacteria play essential roles in plant growth promotion, plant pathogen suppression, and plant immunity boosting. Arabidopsis Col-0 wild-type plants served as the testing ground to assess GD4a's efficacy. Additionally, bacterial enzyme activity and targeted metabolite tests were conducted to validate GD4a's potential for enhancing plant growth and suppressing plant pathogens and diseases. GD4a was subjected to co-incubation with various bacterial, fungal, and oomycete pathogens to evaluate its antagonistic effectiveness in vitro. In vivo pathogen inoculation assays were also carried out to investigate GD4a's role in regulating host plant immunity. Bacterial extracellular exudate (BEE) was extracted, purified, and subjected to untargeted metabolomics analysis. Benzocaine (BEN) from the untargeted metabolomics analysis was selected for further study of its function and related mechanisms in enhancing plant immunity through plant mutant analysis and qRT-PCR analysis. Finally, a comprehensive model was formulated to summarize the potential benefits of applying GD4a in agricultural systems. Our study demonstrates the efficacy of GD4a, isolated from switchgrass, in enhancing plant growth, suppressing plant pathogens and diseases, and bolstering host plant immunity. Importantly, GD4a produces a functional bacterial extracellular exudate (BEE) that significantly disrupts the pathogenicity of B. cinerea by inhibiting fungal conidium germination and hypha formation. Additionally, our study identifies benzocaine (BEN) as a novel small molecule that triggers basal defense, ISR, and SAR responses in Arabidopsis plants. Bacillus amyloliquefaciens strain GD4a can effectively promote plant growth, suppress plant disease, and boost plant immunity through functional BEE production and diverse gene expression.

Keywords: Arabidopsis thaliana; Botrytis cinerea; bacterial extracellular exudates (BEE); benzocaine (BEN); metabolite analysis; systemic resistance.

Figure 1

GD4a promotes plant growth by producing different metabolites and showing diverse enzyme activities. (A) GD4a inoculation increases the growth of Arabidopsis Col-0 seedlings on 1/2 MS media. (B) GD4a inoculation decreases primary root elongation of Arabidopsis Col-0 seedlings on 1/2 MS media. (C) GD4a inoculation increases the fresh-weight (FW) biomass of Arabidopsis Col-0 seedlings on 1/2 MS media. (D) GD4a drenching increases the growth of Arabidopsis Col-0 plants in soil. (E) GD4a drenching increases the rosette width of Arabidopsis Col-0 plants. (F) GD4a drenching increases the leaf chlorophyll content of Arabidopsis Col-0 plants. (G) GD4a drenching increases the shoot FW of Arabidopsis Col-0 plants. (H) GD4a drenching increases the silique production of Arabidopsis Col-0 plants. (I) GD4a drenching increases the seed production of Arabidopsis Col-0 plants. (J) GD4a produces IAA. (K) GD4a produces ammonia. (L) GD4a produces acetoin diacetyl. (M) GD4a produces exopolysaccharides. (N) GD4a produces siderophores. (O) GD4a is positive in pectinase activity. (P) GD4a is positive in amylase activity. (Q) GD4a is positive in ribonuclease activity. (R) GD4a is positive in cellulase activity. (S) GD4a catalases H₂O₂ to O₂. (T) GD4a produces no detectable hydrogen cyanide. (U) GD4a produces no detectable organic acid. (V) GD4a is negative in phosphate solubilization activity. (W) GD4a is negative in ACC deaminase activity. (X) GD4a is negative in chitinase activity.

Figure 2

GD4a is an effective biological control against gray mold disease. (A) GD4a inhibits the growth of the gray mold fungus by the production of bacterial extracellular exudates (BEE). Please note that there is no direct contact between GD4a and B. cinerea, and the BEE is readily soluble in the agar media. (B) Quantification of the fungal growth of the assay in (A). (C) Quantification of the fungal inhibition rate of the assay in (A). (D) GD4a BEE leads to a pathogenicity loss of B. cinerea in Arabidopsis Col-0 plants. The mycelial discs for fungal infection were obtained from the assay in (A). (E) Quantification of the fungal disease symptom of the mycelial disc infection assay in (D). (F) GD4a BEE leads to a pathogenicity loss of B. cinerea in tomato M82 plants. The mycelial discs for fungal infection were obtained from the assay in (A). (G) Quantification of the fungal disease symptom of the mycelial disc infection assay in (F). (H) GD4a BEE leads to a pathogenicity loss of B. cinerea in Arabidopsis Col-0 plants. The fungal infection inoculum was a mix of fungal conidia with BEE of Control/GD4a/D747. (I) Quantification of the fungal disease symptom of the fungal conidium infection assay in (H). (J) GD4a BEE leads to a pathogenicity loss of B. cinerea in tomato M82 plants. The fungal infection inoculum was a mix of fungal conidia with BEE of Control/GD4a/D747. (K) Quantification of the fungal disease symptom of the fungal conidium infection assay in (J). (L) GD4a BEE inhibits the fungal conidium germination and hyphal formation of B. cinerea. HT-BEE, heat-treated BEE (65 °C for 20 min). NT-BEE, nontreated BEE. Representative conidia were pinpointed by black star icons. (M) Illustration of the assay to evaluate GD4a’s biological control against B. cinerea due to the production of volatile bacterial extracellular exudates (VBEE). Created with BioRender.com. (N) GD4a VBEE (VOCs) block the fungal growth of B. cinerea. (O) Quantification of the fungal growth of the assay in (N). (P) Quantification of the fungal inhibition rate of the assay in (N). (Q) Illustration of the assay to evaluate GD4a protecting post-harvested tomato fruits against B. cinerea by VBEE. Created with BioRender.com. (R) GD4a VBEE protects post-harvested tomato fruits from the gray mold. (S) Quantification of the gray mold incidence of the fungal conidium infection assay in (R).

Figure 3

GD4a is an effective biological control against vascular wilt disease, fusarium head blight disease, rice blast disease, fruit rot disease, damping off disease, and bacterial speck disease. (A) GD4a inhibits the growth of F. oxysporum in vitro. (B) Quantification of the growth of F. oxysporum in (A). (C) Quantification of the inhibition rate of F. oxysporum in (A). (D) GD4a inhibits the growth of F. graminearum in vitro. (E) Quantification of the growth of F. graminearum in (D). (F) Quantification of the inhibition rate of F. graminearum in (D). (G) GD4a inhibits the growth of M. oryzae in vitro. (H) Quantification of the growth of M. oryzae in (G). (I) Quantification of the inhibition rate of M. oryzae in (G). (J) GD4a inhibits the growth of P. capsici in vitro. (K) Quantification of the growth of P. capsici in (J). (L) Quantification of the inhibition rate of P. capsici in (J). (M) GD4a inhibits the growth of P. irregulare in vitro. (N) Quantification of the growth of P. irregulare in (M). (O) Quantification of the inhibition rate of P. irregulare in (M). (P) GD4a inhibits the growth of Pst DC3000 in vitro. (Q) Quantification of the inhibition zone of Pst DC3000 in (P). Please note that there is no direct contact between GD4a and the corresponding plant pathogen. The GD4a BEE is readily soluble in the agar media.

Figure 4

GD4a activates ISR against fungal and bacterial pathogens, and GD4a BEE activates SAR against fungal and bacterial pathogens. (A) GD4a root drenching protects Arabidopsis Col-0 plants against the gray mold fungus B. cinerea. (B) Quantification of the fungal disease symptom of the assay in (A). (C) GD4a root drenching protects strawberry Monterey plants against the gray mold fungus B. cinerea. (D) Quantification of the fungal disease symptom of the assay in (C). (E) GD4a root drenching protects Arabidopsis Col-0 plants against the bacterial pathogen Pst DC3000. (F) GD4a local leaf infiltration activates SAR against the bacterial pathogen Pst DC3000. (G) Local leaf infiltration of GD4a BEE activates SAR against the bacterial pathogen Pst DC3000. (H) GD4a BEE infiltration induces ROS production in Arabidopsis Col-0 plants. (I) GD4a BEE induces ROS burst of leaf discs in Arabidopsis Col-0 plants. (J) Quantification of the ROS burst of the assay in (I). (K) GD4a BEE infiltration induces callose deposition in Arabidopsis Col-0 leaves. Representative callose was pinpointed by white star icons. (L) Quantification of the callose deposition in (K).

Figure 5

Untargeted metabolomics analysis of GD4a BEE. (A) GD4a BEE inhibits the gray mold fungus growth in vitro. (B) Quantification of the fungal growth inhibition of the assay in (A) as of surface deployment of alive GD4a cells. (C) Quantification of the fungal growth inhibition of the assay in (A) as of the Oxford cup deployment of alive GD4a cells. (D) Quantification of the fungal growth inhibition of the assay in (A) as of the Oxford cup deployment of fresh 1× raw BEE of GD4a. (E) Quantification of the fungal growth inhibition of the assay in (A) as of the Oxford cup deployment of fresh 10× raw BEE of GD4a. (F) Quantification of the fungal growth inhibition of the assay in (A) as of the Oxford cup deployment of 5-day-old 10× raw BEE of GD4a. (G) Quantification of the fungal growth inhibition of the assay in (A) as of the Oxford cup deployment of fresh 10× raw BEE of GD4a after running through a 0.22 µm filter. (H) Quantification of the fungal growth inhibition of the assay in (A) as of the Oxford cup deployment of fresh 10× refined BEE of GD4a after running through a 0.22 µm filter.

Figure 6

BEN (C9H11NO2) confers resistance to bacterial and fungal pathogens. (A) The 2D structure of BEN. (B) The 3D structure of BEN. (C) BEN shows no antimicrobial activity to bacterial pathogen PstDC3000. (D) BEN shows no antimicrobial activity to fungal pathogen B. cinerea B05.10. (E) BEN-induced resistance against PstDC3000 is concentration-dependent. (F) BEN-induced resistance against B. cinerea B05.10 at different concentrations. (G) BEN drenching-induced resistance against PstDC3000. (H) BEN drenching-induced resistance against B. cinerea B05.10.

Figure 7

Exogenous BEN-induced local defense response in Arabidopsis. (A) The SA signaling pathway is required for BEN-mediated local plant immunity. (B) The JA signaling pathway is necessary for BEN-facilitated local plant immunity. (C) ACP4 and DIR1, which are involved in the fatty acid and lipid signaling pathway, are vital for BEN-interceded local plant immunity. (D) GLR3.2A and GLR3.6A may be involved in BEN-mediated local plant immunity.

Figure 8

The exogenous BEN-induced systemic defense response in Arabidopsis. (A) The SA signaling pathway is required for BEN-mediated systemic plant immunity. (B) The JA signaling pathway is necessary for BEN-facilitated systemic plant immunity. (C) The ACP4, AZI1, and LTP2 genes involved in fatty acid and lipid signaling are vital for BEN-interceded systemic plant immunity. (D) The chemical receptor pathway may be involved in BEN-mediated systemic plant immunity.

Figure 9

Exogenous BEN-induced plant defense/development gene expression in Arabidopsis. (A) NPR1/UBQ5. (B) SID2/UBQ5. (C) PR1/UBQ5. (D) PR2/UBQ5. (E) PR5/UBQ5. (F) JAR1/UBQ5. (G) MYC2/UBQ5. (H) PDF1.2/UBQ5. (I) ACP4/UBQ5. (J) AZI1/UBQ5. (K) LECRK-V1.2/UBQ5. (L) RBOHD/UBQ5. 5hpi-L, locally BEN-infiltrated leaves were collected for total RNA extraction at 5 h post-infiltration. 48hpi-L, locally BEN-infiltrated leaves were collected for total RNA extraction at 48 h post-infiltration. 48hpi-S, systemic non-infiltrated leaves were collected for total RNA extraction at 48 h post-infiltration of BEN to local leaves.

Figure 10

A proposed model outlining how GD4a contributes to safeguarding plant fitness. GD4a can function as a plant growth promoter, a plant immunity activator, and a plant pathogen biological controller. Specifically, GD4a effectively controls the gray mold fungus B. cinerea by producing functional metabolites that directly and indirectly (ISR and SAR) inhibit the fungal conidial germination and hyphae formation and thus provide excellent protection for plants during the growth season and the post-harvest stage, such as storage and transportation processes.