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Mechanism of a Volatile Organic Compound (6-Methyl-2-Heptanone) Emitted From Bacillus subtilis ZD01 Against Alternaria solani in Potato.

ABSTRACT: 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.

SUBMITTER: Zhang D

PROVIDER: S-EPMC8793485 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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: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:BackgroundAlternaria solani (A. solani), the main pathogen of potato early blight, causes serious yield reductions every year. The application of fungicides is the most common and effective method of controlling Alternaria-caused diseases. The differentially expressed transcripts of A. solani infecting potato were identified, revealing a group of valuable candidate genes for a systematic analysis to increase the understanding of the molecular pathogenesis of A. solani, and providing scientific data for formulating additional measures to prevent and control potato early blight. In this study, a deep RNA-sequencing approach was applied to gain insights into A. solani pathogenesis. At 3, 4, and 5 days post inoculation (dpi), RNA samples from the susceptible potato cultivar Favorita infected with A. solani strain HWC-168, were sequenced and utilized for transcriptome analysis, and compared to the transcriptome obtained 0 dpi.ResultsA total of 4430 (2167 upregulated, 2263 downregulated), 4736 (2312 upregulated, 2424 downregulated), and 5043 (2411 upregulated, 2632 downregulated) genes were differentially expressed 3, 4 and 5 dpi, respectively, compared with genes analysed at 0 dpi. KEGG enrichment analysis showed that genes involved in the pathways of amino acid metabolism, glucose metabolism, and enzyme activity were significantly differentially expressed at the late infection stage. Correspondingly, symptoms developed rapidly during the late stage of A. solani infection. In addition, a short time-series expression miner (STEM) assay was performed to analyse the gene expression patterns of A. solani and Profile 17 and 19 showed significant change trends 3, 4 and 5 dpi. Both profiles, but especially Profile 17, included enzymes, including transferases, oxidoreductases, hydrolases and carbohydrate-active enzymes (CAZYmes), which may play important roles in late fungal infection. Furthermore, possible candidate effectors were identified through the adopted pipelines, with 137 differentially expressed small secreted proteins identified, including some enzymes and proteins with unknown functions.ConclusionsCollectively, the data presented in this study show that amino acid metabolism, and glucose metabolism pathways, and specific pathway-related enzymes may be key putative pathogenic factors, and play important roles in late stage A. solani infection. These results contribute to a broader base of knowledge of A. solani pathogenesis in potato, as indicated by the transcriptional level analysis, and provide clues for determining the effectors of A. solani infection.

Project description:BackgroundEarly blight, caused by the necrotrophic fungal pathogen Alternaria solani, is an economically important disease affecting the tuber yield worldwide. The disease is mainly controlled by chemical plant protection agents. However, over-using these chemicals can lead to the evolution of resistant A. solani strains and is environmentally hazardous. Identifying genetic disease resistance factors is crucial for the sustainable management of early blight but little effort has been diverted in this direction. Therefore, we carried out transcriptome sequencing of the A. solani interaction with different potato cultivars with varying levels of early blight resistance to identify key host genes and pathways in a cultivar-specific manner.ResultsIn this study, we have captured transcriptomes from three different potato cultivars with varying susceptibility to A. solani, namely Magnum Bonum, Désirée, and Kuras, at 18 and 36 h post-infection. We identified many differentially expressed genes (DEGs) between these cultivars, and the number of DEGs increased with susceptibility and infection time. There were 649 transcripts commonly expressed between the potato cultivars and time points, of which 627 and 22 were up- and down-regulated, respectively. Interestingly, overall the up-regulated DEGs were twice in number as compared to down-regulated ones in all the potato cultivars and time points, except Kuras at 36 h post-inoculation. In general, transcription factor families WRKY, ERF, bHLH, MYB, and C2H2 were highly enriched DEGs, of which a significant number were up-regulated. The majority of the key transcripts involved in the jasmonic acid and ethylene biosynthesis pathways were highly up-regulated. Many transcripts involved in the mevalonate (MVA) pathway, isoprenyl-PP, and terpene biosynthesis were also up-regulated across the potato cultivars and time points. Compared to Magnum Bonum and Désirée, multiple components of the photosynthesis machinery, starch biosynthesis and degradation pathway were down-regulated in the most susceptible potato cultivar, Kuras.ConclusionsTranscriptome sequencing identified many differentially expressed genes and pathways, thereby contributing to the improved understanding of the interaction between the potato host and A. solani. The transcription factors identified are attractive targets for genetic modification to improve potato resistance against early blight. The results provide important insights into the molecular events at the early stages of disease development, help to shorten the knowledge gap, and support potato breeding programs for improved early blight disease resistance.

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Mechanism of a Volatile Organic Compound (6-Methyl-2-Heptanone) Emitted From Bacillus subtilis ZD01 Against Alternaria solani in Potato.

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