Unlocking antagonistic potential of Bacillus amyloliquefaciens KRS005 to control gray mold

Abstract

To establish a safe, efficient, and simple biocontrol measure for gray mold disease caused by Botrytis cinerea, the basic characteristics and antifungal activity of KRS005 were studied from multiple aspects including morphological observation, multilocus sequence analysis and typing (MLSA-MLST), physical-biochemical assays, broad-spectrum inhibitory activities, control efficiency of gray mold, and determination of plant immunity. The strain KRS005, identified as Bacillus amyloliquefaciens, demonstrated broad-spectrum inhibitory activities against various pathogenic fungi by dual confrontation culture assays, of which the inhibition rate of B. cinerea was up to 90.3%. Notably, through the evaluation of control efficiency, it was found that KRS005 fermentation broth could effectively control the occurrence of tobacco leaves gray mold by determining the lesion diameter and biomass of B. cinerea on tobacco leaves still had a high control effect after dilution of 100 folds. Meanwhile, KRS005 fermentation broth had no impact on the mesophyll tissue of tobacco leaves. Further studies showed that plant defense-related genes involved in reactive oxygen species (ROS), salicylic acid (SA), and jasmonic acid (JA)-related signal pathways were significantly upregulated when tobacco leaves were sprayed with KRS005 cell-free supernatant. In addition, KRS005 could inhibit cell membrane damage and increase the permeability of B. cinerea. Overall, KRS005, as a promising biocontrol agent, would likely serve as an alternative to chemical fungicides to control gray mold.

Keywords: Bacillus amyloliquefaciens; Botrytis cinerea; biological control; gray mold; plant immunity.

Figure 1

Identify the taxonomy of an endophyte microbe KRS005 from cotton stem. (A) Morphological characteristics of strain KRS005 on LB medium plate cultured 12 and 24 h. (B) Gram staining observation under an optical microscope, Pseudomonas alcaligenes was severed as gram-negative control. (C) Phylogenetic tree of the KRS005 strain and its homolog bacterial species by searching with the gyrB, gyrA, and rpoB nucleotide sequences. The phylogenetic tree was performed by MLSA-MLST methods using the connection of gyrB, gyrA, and rpoB sequence, with MEGA6.0 software. Escherichia coli (08BKT055439) was used as an outgroup to root the tree. The bootstrap analysis was performed with 10,000 replications.

Figure 2

Physiological and biochemical characteristics of strain KRS005. (A) Nitrogen fixation, phosphate and potassium solubilization ability, and siderophore production of KRS005 were determined. (B) Gelatin liquefaction, amylase activity, proteinase activity, and indole assays. (C) Sulfide reaction, citrate, methyl red, and nitrate reduction were determined. E. coli DH5α was severed as control. “+” indicates that the result is positive reaction, “−” indicates that the result is negative reaction. In all of the physiological and biochemical tests, Escherichia coli DH5α was used as a control, each treatment was conducted in three replicates, and every test was performed at least three times.

Figure 3

The broad-spectrum activity of KRS005 against plant pathogenic fungi. (A) In vitro effect of KRS005 on the plant pathogens. Botrytis cinerea and Verticillium dahliae, Fusarium graminearum, Colletotrichum falcatum, Colletotrichum gloeosporioides, and Magnaporthe oryzae were cultured on potato dextrose agar (PDA) medium in the presence of KRS005. (B) The inhibition rate of KRS005 against plant pathogenic fungi. (C) Phenotypes for KRS005 cell-free supernatant against plant pathogenic fungi. (D) The inhibition rate of KRS005 cell-free supernatant against plant pathogenic fungi. Blue text indicates the inhibition rate. Error bars represent standard deviations. *, ***, and **** indicate the significant difference at p < 0.05, p < 0.001, and p < 0.0001, respectively, according to the unpaired Student’s t-test.

Figure 4

Effect of different treatments of KRS005 on gray mold. (A) Antifungal effect of different KRS005 culture filtrate on Botrytis cinerea growth. (B) Different KRS005 culture filtrate inhibits gray mold lesions on tobacco leaves at 4 days post-inoculation (dpi). LB: LB broth, 100 × FB: diluted 100-fold of fermentation broth, 100 × FL: diluted 100-fold of fragmentized liquid, Supernatant: cell-free supernatant. (C) Disease incidences of infected leaves at 4 dpi. The diameter of the lesion was decreased in KRS005-treated leaves compared with the control. The grade of disease was classified as 0, 1, 2, with the lesion diameter as 0 mm, 1~10 mm, and more than 10 mm, respectively. (D) The fungal biomass in tobacco leaves by quantitative PCR (qPCR) at 4 dpi. Error bars represent standard deviations, and asterisks *, **, ***, and **** indicate the significant difference at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively, according to unpaired Student’s t-test.

Figure 5

KRS005 triggers plant immunity response. (A) Phytotoxicity of KRS005 culture filtrate on tobacco leaves after 12 h (Scale bars = 50 μm). (B,C) DAB staining observations of tobacco leaves treated with KRS005 cell-free supernatant. LB-OP, Luria-Bertani optimized medium optical density; GFP, Green fluorescent protein used for negative control; EG1, effector VdEG1 that triggers cell death (Scale bars = 10 μm). (D) DAB optical density (OD) data. (E,F) Expression of defense-related genes in tobacco leaves of uninoculated or uninoculated cell-free supernatant-treated or mock-treated tobacco plants 6 h after treatments. Error bars represent standard deviations, and asterisks *, ***, and **** indicate the significant difference at p < 0.05, p < 0.001, and p < 0.0001, respectively, according to unpaired Student’s t-test.

Figure 6

The KRS005 influences the growth and morphology of Botrytis cinerea. (A) The cell-free supernatant of KRS005 significantly inhibited the mycelial growth of B. cinerea. LB-OP: PDA medium was mixed with optimized Luria-Bertani at 2:1, FB: PDA medium was mixed with fermentation broth at 2:1, Supernatant: PDA medium was mixed with fermentation broth at 2:1. (B) Botrytis cinerea colony diameter from three repeated experiments. (C) Inhibition of the plate fungistatic experiment of the normal mycelium, and mycelium treated with 30% of cell-free supernatant. LB medium as negative control. S: cell-free supernatant. The fungal structures were observed with 10 × and 40 × objectives, respectively (Scale bars = 20 μm). (D) Effect of KRS005 cell-free supernatant on cell membrane permeability of B. cinerea. Error bars represent standard deviations. The asterisks *, ***, and **** indicate the significant difference at p < 0.05, p < 0.001, and p < 0.0001, respectively, according to the unpaired Student’s t-test.