The Lethal Effect of <i>Bacillus subtilis</i> Z15 Secondary Metabolites on <i>Verticillium dahliae</i>

Leaf, stem, and stripe rusts are the most important diseases of wheat worldwide. In southern Sonora, Mexico, leaf rust is endemic and has caused serious epiphytotics; proper control relies on breeding for resistance and fungicide applications. The extensive utilization of chemicals to control diseases of plants, the emergence of resistant phytopathogens to fungicides, and the damage to the health of producers and consumers, has promoted the search for viable alternatives that guaranty a sustainable agriculture production, minimizing the impact on the environment. In this work, the biofungicides Roya Out® and Best Ultra®F were evaluated for control of leaf rust on cultivar CIRNO C2008, under a randomized complete block experimental design with three replications. After one application of the biofungicides and the inoculation with a urediniospore suspension, two other applications were carried out. Disease severity was evaluated following Cobb´s modified scale and the analysis of variance was performed with SAS, and the mean comparison with Duncan´s multiple range test (α = 0.05). Other variables measured were: spike length and weight, number of grains per spike, grain length and weight, a thousand grain weight, and grain yield per plot. The disease showed up during the soft dough stage and disease severity was 32% for plots treated with Best Ultra®F, 27% with Roya Out®, and 25% in the untreated check plots. Despite the infection, the highest grain yield estimated was obtained from plots treated with Roya Out® (7.22 t ha-1), followed by Best Ultra®F (7.03), and the untreated check (6.12 t ha-1).

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“…[42], Rhizoctonia solani Kühn [43], Sclerotinia spp. [44], Verticillium dahliae Kleb [45], Botrytis cinerea Pers.:Fr. [46], Alternaria spp.…”

Section: Resultsmentioning

confidence: 99%

Evaluation of two bio-fungicides for control of leaf rust (Puccinia triticina Eriks.) on durum wheat cultivar CIRNO C 2008

Rosas-Jáuregui¹,

Fuentes-Dávila²,

Félix-Fuentes³

et al. 2022

World J. Adv. Res. Rev.

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“…Previous studies showed that both BS-Z15 and its fermentation broth possess the strong anti-VD-991 activity [ 14 , 15 ], indicating that the active compound from BS-Z15 is water-soluble and extracellular-secreted, indicating a basic purification strategy. Therefore, HCl precipitation was first performed to concentrate the compound.…”

Section: Discussionmentioning

confidence: 99%

“…The anti-VD-991 activity was detected by the agar-disk diffusion assay [ 15 ]. VD-991 were cultured in Czapek–Dox Medium at 25°C, 180 rpm for 24 h, and 20 μL of spore suspension of VD-991 (10^5 cfu/mL) were coated on Czapek–Dox Agar Medium.…”

Section: Methodsmentioning

confidence: 99%

“…Over the last couple of decades, biological control has attracted more attention, because of its advantages include low toxicity, low pollution, and efficiency, and numerous Bacillus species are widely used for biocontrolling a broad range of fungal plant pathogens [ 11 – 13 ]. Prior studies showed that one Bacillus subtilis strain Z15 (BS-Z15), isolated from cotton field of Xinjiang, China, had excellent antagonistic effect on VD-991, providing a positive role on controlling cotton verticillium wilt in China [ 14 , 15 ]. However, the chemical substance produced by BS-Z15 for resistance to VD-991 is still unknown.…”

Section: Introductionmentioning

confidence: 99%

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

Lin

Zhang

et al. 2022

PLoS ONE

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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.

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“…Then, circular bacterial blocks with a diameter of 8 mm were inoculated in the centre of Czapek–Dox agar plates containing different concentrations of C17 mycosubtilin (0, 1, 3, 10 μg/mL, of which 0 μg/mL was the control group and the other concentrations were the experimental group) and cultured at 25 °C. On the 3rd, 6th, 12th, and 18th days, the colony diameters were measured using ImageJ software (Version 1.51j8) by the criss-cross method [ 43 ], and the growth inhibition degree of C17 mycosubtilin to Vd 991 was calculated according to the following formula: inhibition rate (%) = [(colony diameter of control group-colony diameter of experimental group)/colony diameter of control group] × 100%.…”

Section: Methodsmentioning

confidence: 99%

Transcriptome Analysis Reveals That C17 Mycosubtilin Antagonizes Verticillium dahliae by Interfering with Multiple Functional Pathways of Fungi

Zhang

Lin

Yang

et al. 2023

Biology

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Verticillium wilt is a kind of soil-borne plant fungal disease caused by Verticillium dahliae (Vd). Vd 991 is a strong pathogen causing cotton Verticillium wilt. Previously, we isolated a compound from the secondary metabolites of Bacillus subtilis J15 (BS J15), which showed a significant control effect on cotton Verticillium wilt and was identified as C17 mycosubtilin. However, the specific fungistatic mechanism by which C17 mycosubtilin antagonizes Vd 991 is not clear. Here, we first showed that C17 mycosubtilin inhibits the growth of Vd 991 and affects germination of spores at the minimum inhibitory concentration (MIC). Morphological observation showed that C17 mycosubtilin treatment caused shrinking, sinking, and even damage to spores; the hyphae became twisted and rough, the surface was sunken, and the contents were unevenly distributed, resulting in thinning and damage to the cell membrane and cell wall and swelling of mitochondria of fungi. Flow cytometry analysis with ANNEXINV-FITC/PI staining showed that C17 mycosubtilin induces necrosis of Vd 991 cells in a time-dependent manner. Differential transcription analysis showed that C17 mycosubtilin at a semi-inhibitory concentration (IC50) treated Vd 991 for 2 and 6 h and inhibited fungal growth mainly by destroying synthesis of the fungal cell membrane and cell wall, inhibiting its DNA replication and transcriptional translation process, blocking its cell cycle, destroying fungal energy and substance metabolism, and disrupting the redox process of fungi. These results directly showed the mechanism by which C17 mycosubtilin antagonizes Vd 991, providing clues for the mechanism of action of lipopeptides and useful information for development of more effective antimicrobials.

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The Lethal Effect of Bacillus subtilis Z15 Secondary Metabolites on Verticillium dahliae

Cited by 5 publications

References 37 publications

Evaluation of two bio-fungicides for control of leaf rust (Puccinia triticina Eriks.) on durum wheat cultivar CIRNO C 2008

Evaluation of two bio-fungicides for control of leaf rust (Puccinia triticina Eriks.) on durum wheat cultivar CIRNO C 2008

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

Transcriptome Analysis Reveals That C17 Mycosubtilin Antagonizes Verticillium dahliae by Interfering with Multiple Functional Pathways of Fungi

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