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Antifungal Effects of Volatiles Produced by Bacillus subtilis Against Alternaria solani in Potato.

ABSTRACT: Antifungal activities of plant-beneficial Bacillus have been widely studied in recent years. Numerous studies have studied the antifungal mechanisms of soluble non-volatile bioactive compounds such as lipopeptides and proteins produced by Bacillus against soil-borne diseases. However, the antagonistic mechanisms of volatile organic compounds (VOCs) from Bacillus against airborne phytopathogens are still largely unknown, and the function of Alternaria solani pathogenic genes has not been well identified. Here, we first isolated a Bacillus strain with strong antifungal activity and finally identified it as B. subtilis ZD01. Then, the antagonistic mechanisms of VOCs produced by strain ZD01, against A. solani, an airborne fungal pathogen that can cause early blight diseases of potato, were studied. We showed that VOCs produced by strain ZD01 can reduce the colony size and mycelial penetration and can cause serious morphological changes of A. solani. Scanning electron microscope (SEM) observation showed that VOCs released by ZD01 could cause more flaccid and gapped hyphae of A. solani. Also, we found that VOCs produced by ZD01 can inhibit the conidia germination and reduce the lesion areas and number of A. solani in vivo significantly. Meanwhile, based on gas chromatography/mass spectrometry (GC/MS) analysis, 29 volatile compounds produced by strain ZD01 were identified. Out of 29 identified VOCs, 9 VOCs showed complete growth inhibition activities against A. solani. Moreover, we identified two virulence-associated genes (slt2 and sod) in A. solani. slt2 is a key gene that regulates the mycelial growth, penetration, sporulation, and virulence in vivo in A. solani. In addition, sod plays a significant role in the SOD synthetic pathway in A. solani. Results from qRT-PCR showed that the transcriptional expression of these two genes was down-regulated after being treated by VOCs produced by ZD01. These results are useful for a better understanding of the biocontrol mechanism of Bacillus and offer a potential method for potato early blight disease control.

SUBMITTER: Zhang D

PROVIDER: S-EPMC7311636 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Antifungal Effects of Volatiles Produced by Bacillus subtilis Against Alternaria solani in Potato.

Zhang Dai D Yu Shuiqing S Yang Yiqing Y Zhang Jinglin J Zhao Dongmei D Pan Yang Y Fan Shasha S Yang Zhihui Z Zhu Jiehua J

Frontiers in microbiology 20200617

Antifungal activities of plant-beneficial Bacillus have been widely studied in recent years. Numerous studies have studied the antifungal mechanisms of soluble non-volatile bioactive compounds such as lipopeptides and proteins produced by Bacillus against soil-borne diseases. However, the antagonistic mechanisms of volatile organic compounds (VOCs) from Bacillus against airborne phytopathogens are still largely unknown, and the function of Alternaria solani pathogenic ...[more]

PMID: 32625175

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Project description:Bacterial biocontrol agent/s (BCAs) against plant diseases are eco-friendly and sustainable options for profitable agricultural crop production. Specific beneficial strains of Bacillus subtilis are effective in controlling many fungal diseases including Alternaria blight caused by a notorious pathogen "Alternaria solani". In the present study, the biocontrol attributes of a newfangled strain of B. subtilis (BS-01) have been investigated and its bioactive compounds were also identified against A. solani. The volatile organic compounds (VOCs) produced by BS-01 in organic solvents viz., n-hexane, dichloromethane, and ethyl acetate were extracted and their antifungal efficacy has evaluated against A. solani. Also, the preventive and curative biocontrol method to reduce the fungal load of A. solani was estimated by both foliar and seed applications on infected tomato (Solanum lycopersicum) plants as determined by quantitative PCR assays. Growth chamber bioassay revealed that both foliar and seed application of BS-01 on tomato plants previously or subsequently infected by A. solani significantly reduced the pathogen load on inoculated tomato foliage. Results showed that antifungal bioassays with various concentrations (10-100 mg mL-1) of extracted metabolites produced by BS-01 in ethyl acetate fraction showed the highest inhibition in fungal biomass (extracellular metabolites: 69-98% and intracellular metabolites: 48-85%) followed by n-hexane (extracellular metabolites: 63-88% and intracellular metabolites: 35-62%) and dichloromethane (extracellular metabolites: 41-74% and intracellular metabolites: 42-70%), respectively. The extracted volatile compounds of BS-01 were identified via GC-MS analysis and were found in great proportions in the organic fractions as major potent antifungal constituents including triphenylphosphine oxide; pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl); pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(phenylmethyl); n-hexadecanoic acid; n-tridecan-1-ol; octadecane; octadecanoic acid; eicosane and dodecyl acrylate. Separate or mixture of these bioactive VOCs had the potential to mitigate the tomato early blight disease severity in the field that would act as a sustainable plant protection strategy to generate profitable tomato production.

Project description:Alternaria solani is an airborne fungus and the primary causal agent of potato early blight worldwide. No available fungicides that are both effective and environmentally friendly are usable to control this fungus. Therefore, biological control is a potential approach for its suppression. In this study, Bacillus subtilis strain ZD01's fermentation broth strongly reduced A. solani pathogenicity under greenhouse conditions. The effects of strain ZD01's secondary metabolites on A. solani were investigated. The exposure of A. solani hyphae to the supernatant resulted in swelling and swollen sacs, and the ZD01 supernatant reduced A. solani conidial germination significantly. Matrix-assisted laser desorption/ionization time of flight mass spectrometry and pure product tests revealed that fengycins were the main antifungal lipopeptide substances. To elucidate the molecular mechanism of the fengycins' biological control, RNA sequencing analyses were performed. A transcriptome analysis revealed that 304 and 522 genes in A. solani were differentially expressed after 2-h and 6-h fengycin treatments, respectively. These genes were respectively mapped to 53 and 57 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In addition, the most enriched KEGG pathway analysis indicated that the inhibitory mechanisms of fengycins against A. solani regulated the expression of genes related to cell wall, cell membrane, transport, energy process, protein synthesis and genetic information. In particular, cell wall and cell membrane metabolism were the main processes affected by fengycin stress. Scanning and transmission electron microscope results revealed hyphal enlargement and a wide range of abnormalities in A. solani cells after exposure to fengycins. Furthermore, fengycins induced chitin synthesis in treated cells, and also caused the capture of cellular fluorescent green labeling and the release of adenosine triphosphate (ATP) from outer membranes of A. solani cells, which may enhance the fengycins ability to alter cell membrane permeability. Thus, this study increases the transcriptome data resources available and supplies a molecular framework for B. subtilis ZD01 inhibition of A. solani HWC-168 through various mechanisms, especially damaging A. solani cell walls and membranes. The transcriptomic insights may lead to an effective control strategy for potato early blight.

Project description:The antagonistic mechanisms of soluble non-volatile bioactive compounds, such as proteins and lipopeptides emitted from Bacillus have been widely studied. However, there are limited studies on the antifungal mechanisms of volatile organic compounds (VOCs) produced by Bacillus against plant fungal diseases. In this study, the antagonistic mechanisms of one specific VOC, 6-methyl-2-heptanone, against Alternaria solani were investigated. To optimize the extraction conditions of headspace solid-phase microextraction, a 50/30-μm divinylbenzene/carboxen/polydimethylsiloxane fiber at 50°C for 40 min was used. For gas chromatography-mass spectrometry using a free fatty acid phase capillary column, 6-methyl-2-heptanone accounted for the highest content, at 22.27%, of the total VOCs from Bacillus subtilis ZD01, which inhibited A. solani mycelial growth strongly in vitro. Therefore, 6-methyl-2-heptanone was selected as the main active chemical to elucidate the action mechanisms against A. solani. Scanning and transmission electron microscopy analyses revealed that after exposure to an EC50 dose of 6-methyl-2-heptanone, A. solani hyphal cells had a wide range of abnormalities. 6-Methyl-2-heptanone also caused the capture of cellular fluorescent green label and the release of adenosine triphosphate (ATP) from outer membranes A. solani cells, which may enhance 6-methyl-2-heptanone ability to reach the cytoplasmic membrane. In addition, 6-methyl-2-heptanone showed strong inhibitory effect on A. solani conidial germination. It also damaged conidial internal structures, with the treated group having collapsed shrunken small vesicles as observed by transmission electron microscopy. Because 6-methyl-2-heptanone showed strong effects on mycelial integrity and conidial structure, the expression levels of related pathogenic genes in A. solani treated with 6-methyl-2-heptanone were investigated. The qRT-PCR results showed that transcriptional expression levels of slt2 and wetA genes were strongly down-regulated after exposure to 6-methyl-2-heptanone. Finally, because identifying the functions of pathogenic genes will be important for the biological control of A. solani, the wetA gene was identified as a conidia-associated gene that plays roles in regulating sporulation yield and conidial maturation. These findings provide further insights into the mechanisms of VOCs secreted by Bacillus against A. solani.

Dataset Information

Antifungal Effects of Volatiles Produced by Bacillus subtilis Against Alternaria solani in Potato.

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Antifungal Effects of Volatiles Produced by <i>Bacillus subtilis</i> Against <i>Alternaria solani</i> in Potato.

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