| Fusarium graminearum is a plant pathogen that causes Fusarium head blight(FHB)in cereal grains,resulting significant economic losses due to reduction in grain yield and quality,as well as the production of DON mycotoxins that can contaminate food and feed.The DON contamination poses a significant risk to human and animal health.Hence researchers are looking for various ways to control this pathogen and its hazardous mycotoxins to avoid health and economic losses.Prevention and control strategies for FHB and DON mycotoxin contamination include pre-harvest and post-harvest stages which directly impact the quality of the food.Current regulatory preventive strategies can prohibit both the FHB and mycotoxin toxicosis to different capacities.One of the major concerns in the food industry is the occurrence of grains with mycotoxin levels above acceptable limits and the chemical treatment of products intended for human consumption to reduce or eliminate these harmful substances.Moreover,obtaining an economical or commercially feasible method for the detoxification of contaminated grains has been unsuccessful.Hence,researchers are looking for convenient,eco-friendly and cost-effective approaches to address the fungal contamination and inhibition of their mycotoxins.One of the approaches addressing these advantages and is being used nowadays is biological control of fungal pathogens using bacterial strains.In this research,we found a strain of Bacillus genus more specifically identified as Bacillus siamensis Sh420,which was isolated from the soil of a wheat field,can inhibit the growth of a potent pathogenic fungus called F.graminearum.We identified and characterized this strain of bacteria as B.siamensis using 16 S r RNA sequencing technique.The results suggest that this strain of bacteria could be a safer alternative compared to fungicides for the control of fungal pathogens.During in-vitro dual culturing,our isolated strain showed a satisfactory inhibition of F.graminearum.Later it showed a strong antagonistic activity to prevent the infestation of Fusarium in treated wheat grains as compared to untreated grains.In these infected and non-infected grains,DON is quantified using HPLC where surprisingly Sh420 strain inhibited the production of DON to 67.8% as compared to control.While in petri plate DON was inhibited to 61.55% in Sh420 treated plate as compared to the control plate.Furthermore,investigation for the secretion of hydrolytic enzymes(cellulase,protease,glucanase and chitinase)is done using specific substrate culture where the bacteria showed its activity by making clearance halo zone.After confirmation of their activity quantification,spectrophotometer technique were used to show the levels of enzymatic release.Effects of Sh420 on the ultrastructure of F.graminearum were analyzed using scanning electron microscopy(SEM)and transmission electron microscopy(TEM)which showed serious structural deformities in the fungus mycelia and cell structure.SEM confirms that fungal samples treated with the Sh420 strain exhibited shrinking,curling,plasmolysis and twisting in the hyphae.Beside this,TEM showed deformities in the fungal hyphae at the cellular level,which includes loss of cell integrity,damage to the cell membrane,shrinking of the cell,displacement of the cytoplasm,and degeneration of the organelles.On the other hand,the control hyphae retained their normal cellular shape and integrity.From further investigation Indole Acetic Acid(IAA)production is also confirmed in Sh420 which indicates its plant growth promoting characteristics.Later on pot experiment in green house confirms that B.siamensis Sh420 showed plant growth promoting and plant protecting potential against pathogens,where bacterial treated wheat plants were healthy and showed significant growth and no disease symptoms were seen on them.Here,Fusarium treated seedlings showed highest disease severity of 5 out of a maximum scale of 6.During the interaction of Sh420 with Fusarium,overall effect on the transcriptomic profile indicated interesting results in genetic regulation of the phytopathogen by creating regulative alterations in genes related to biological processes,cellular component,and molecular functions.From the differentially expressed genes(DEGs),we observed that cell wall and membrane synthesis-related genes/enzymes like,glucan endo-1,6-beta-glucosidase,two key genes involved in ergosterol pathway Erg1 and Fg PEX4 in treated Fusarium were down-regulated,suggesting increased stress conditions and increase susceptibility and weakness of fungal cell walls resulting loose stability and fluidity of its cell membranes.Fbp1,involved in sexual development,vegetative growth,pigmentation and fungal virulence is suppressed which suggests decline in growth and virulence activity of the pathogen.Furthermore,genes involved in regulation of DON like TRI5 and TRI10 were also observed downregulated,which also indicates suppression in production of mycotoxin,later these DEGs are validated using q RT-PCR.To determine whether this strain has antimicrobial properties,a study used PCR technology to detect specific genes responsible for producing antifungal lipopeptides(LPs)using bacterial DNA.In continuation of this study,we used isolated LPs from B.siamensis and checked its fungicidal potential which showed a strong inhibitory activity against F.graminearum.Later,this crude extract is analyzed using UPLC Q-TOF/MS technique through which we identified important inhibitory compounds like bacillomycin D,iturin C,iturin A3,fengycin A,and three surfactin compounds.LPs extract effects on Fusarium were analyzed using fluorescent microscopy which shows blurred images due to loss of chitin and glucans from the cell walls.LPs creates pores in fungal hyphae by depolarizing membranes,inhibiting chitin and glucan synthases,and inducing apoptosis confirming cell death and damaging effects.Furthermore,LPs of Sh420 can inhibit Fusarium mold invasion in grapes and no disease symptoms or dark spots or lesion were seen in fruits treated with Sh420 LP,while it inhibit the fungus to 78% in grape fruit when inoculated pathogen along with LPs.Additionally,it is found that Sh420 LPs have the potential to activate the antioxidant properties in fruit and protect them from getting rusty/brownish.The greatest increase in antioxidant activity was seen in fruits which were treated with LPs.Due to exposure of Sh420 LPs,the total phenolic content was significantly increased in control and LPs treated fruits.Overall Sh420 strain has biocontrol potential to inhibit the F.graminearum growth and reduce DON contamination. |
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