Isolation and characterization of a mycosubtilin homologue antagonizing Verticillium dahliae produced by Bacillus subtilis strain Z15

Abstract

Bacillus subtilis strain Z15 (BS-Z15) was isolated from the cotton field of Xinjiang, China, and characterized as an effective biocontrol agent antagonizing plant pathogen Verticillium dahliae 991 (VD-991). However, the chemical substance produced by BS-Z15 for resistance to VD-991 remains elusive. Here, a serial purification methods including HCl precipitation, organic solvent extraction, and separation by semi-preparative High-Performance Liquid Chromatography were performed to obtain a single compound about 3.5 mg/L from the fermentation broth of BS-Z15, which has an antifungal activity against VD-991. Moreover, Fourier Transform Infrared spectrum, Nuclear Magnetic Resonance Spectroscopy, and Tandem Mass Spectrometry analyses were carried out to finally confirm that the active compound from BS-Z15 is a mycosubtilin homologue with C17 fatty acid chain. Genomic sequence prediction and PCR verification further showed that the BS-Z15 genome contains the whole mycosubtilin operon comprising four ORFs: fenF, mycA, mycB, and mycC, and the expression levels of mycA-N, mycB-Y and mycC-N reached a peak at 32-h fermentation. Although mycosubtilin homologue at 1 μg/mL promoted the germination of cotton seed, that with high concentration at 10 μg/mL had no significant effect on seed germination, plant height and dry weight. Furthermore, mycosubtilin homologue sprayed at 10 μg/mL on two-week-old cotton leaves promotes the expression of pathogen-associated genes and gossypol accumulation, and greatly decreases VD-991 infection in cotton with disease index statistics. This study provides an efficient purification strategy for mycosubtilin homologue from BS-Z15, which can potentially be used as a biocontrol agent for controlling verticillium wilt in cotton.

The ScAPD1‐like gene from the desert moss Syntrichia caninervis enhances resistance to Verticillium dahliae via phenylpropanoid gene regulation

“…Rehydration was achieved by transferring 24 h dried gametophytes to filter paper saturated with 8 ml distilled water at 25°C for 48 h. For the salt, SA, MeJA, ACC, and ABA treatments, the hydrated gametophores were placed on filter paper saturated with 8 ml 250 m m NaCl, 1 m m SA, 50 μ m MeJA,100 μ m ACC, and 200 μ m ABA solutions for 48 h, respectively; For the cold and heat stress treatments, hydrated gametophores were transferred on filter paper saturated with 8 ml distilled H 2 O and placed into chambers at 4°C and 45°C for 48 h, respectively. The highly virulent V. dahliae fungal strain Vd991 was a kind gift from Professor Huixin Zhao at Xinjiang Normal University (Lin et al., 2022) was used for V. dahliae treatments. Vd991 strain cultured for 7 days on potato dextrose agar medium at 25°C, followed by inoculation into potato dextrose broth medium at 25°C for 7 days on a shaker.…”

Bacillus velezensis TSA32-1 as a Promising Agent for Biocontrol of Plant Pathogenic Fungi

“…myc encodes mycosubtilin, which has an antifungal function. B. subtilis Z15, which produces mycosubtilin, applied to a cotton field confirmed its ability to inhibit wilt disease caused by Verticillium dahlia [ 50 ].…”

Metabolites from Bacillus subtilis J-15 Affect Seedling Growth of Arabidopsis thaliana and Cotton Plants

“…Mycosubtilin, a lipopeptide purified from J-15-Ms, has strong antifungal activity [ 44 ]; however, its effect on plant growth has not been reported. The treatment of Arabidopsis seedlings and cotton with different mycosubtilin concentrations showed the same pattern of pro-growth at lower concentrations and inhibition at higher concentrations.…”

“…Bacillus subtilis J-15 was activated by scribing, and single colonies were picked and cultured in beef paste peptone medium at 37 °C with shaking at 180 revolutions/min for 18 h. The J-15-Ms were extracted with acetone, chloroform (to remove the heteroproteins), and n-butanol [ 83 ]. The J-15-Ms was freeze-dried and solubilized using dimethyl sulfoxide, then purified by high-performance liquid chromatography to obtain the lipopeptide mycosubtilin [ 44 ].…”

“…The previous analysis of the J-15 genome found that the major antifungal gene clusters reported in Bacillus subtilis only contained the complete mycosubtilin and bacilibactin manipulators, suggesting that the two could be the major antifungal active substances [ 43 ]. Another study also isolated and purified the J-15-Ms and identified the antagonist as mycosubtilin [ 44 ]. Based on this, we investigate the mycosubtilin plant growth regulatory mechanism and provide a theoretical basis for applying the J-15 strain in plant growth improvement.…”