| Potato early blight is a worldwide fungal disease caused by Alternaria solani,with the rate of diseased leaves up to 50%to 70%in severely areas.Nowadays,chemical fungicides are the most effective agents to control early blight.However,the indiscriminate using and even abuse of chemical fungicides have already caused the appearance of resistant pathogens,which will further threaten the food safety and human health.Due to this,there is an increasing need for alternative environmentally friendly effective methods to control potato early disease.Bacillus species have been widely used in the biological control of plant pathogens owing to their strong inhibitory activities against various pathogenic fungi.In this study,The highefficiency biocontrol isolates against A.solani were screened in vitro and vivo assays.The antagonistic mechanism of secondary metabolism produce by Bacillus strains is also investigated.This study will help provide strain resources and new approaches for the biological control of A.solani.The main results of this study are as follows:1.B.subtilis ZD01 effectively controlled potato early blight disease.A total of 213 isolated Bacillus strains were obtained from rhizosphere of potato in 7 different areas in China through plate confrontation method and greenhouse test.Strain ZD01 exhibited the most preventive effects against A.solani.B.subtilis ZD01 could reduce the degree of leaf yellowing,and maintain the chlorophyll content and photosynthetic performance of the leaves after infection by A.solani.The chlorophyll content of leaves spraying with 5×103 CFU/mL、5×105 CFU/mL and 5×107 CFU/mL of strain ZD01 fermentation broth increased by 33.33%,53.70%,and 59.40%compared to the control group,respectively.The fluorescence parameter images of the treatment groups were uniform without low dark areas,and ZD01 fermentation could improve the fluorescence intensity of OJIP curve.Chlorophyll fluorescence parameters including F0、Fm、Fv/Fm and qN in treated leaves changed compared with control group.These results showed that B.subtilis ZD01 can improve the electron acceptor performance of PSII reaction center of leaves.Based on the genome sequence,a total of 3944 protein coding genes were predicted,and a total of 3656 genes were annotated by COG,accounting for 82.05%of ZD01 genome sequence.The KEGG database analysis showed that 227 pathways were annotated including the biosynthesis of phenazines,alkenes and streptomycin,with the ability to produce a variety of secondary metabolites.Combining morphology and 31 housekeeping genes,ZD01 was identified as Bacillus subtilis.2.Fengycins were the main active ingredient of antifungal lipopeptides secreted by B.subtilis ZD01against A.solani,which can interfere with the biosynthesis of cell wall and membrane of A.solani.In the present research,we found that the metabolites of B.subtilis ZD01 showed the same effect on mycelial growth,conidial germination and potato early blight disease controlling as fermentation broth.To find the active compound in ZD01 metabolites,the whole genome sequence of ZD01 was obtained.The result revealed that strain ZD01 contained 10 secondary metabolism gene clusters.Through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS),fengycins were identified as the main active component in lipopeptides,with a MIC value of 3.91 μg/mL.In order to further explore its antifungal mechanism,transcriptome analysis was conducted on the hyphae of A.solani HWC-168 treated with fengycins.The result showed that fengycins significantly inhibited the nucleic acid and energy metabolism processes of A.solani.While SNF2,GAPDH,LYS1 and other related genes are significantly downregulated,and the differentially expressed genes in cell wall and membrane components are significantly upregulated involved in chitin and ergosterol metabolism processes such as CHS1,AOC3,ERG4,and ERG3.Phenotypic analysis confirmed that fengycins could lead to abnormal structure of hyphae,cell membrane rupture and formation of holes,and leakage of intracellular substances of A.solani.The content of chitin and ergosterol in A.solani mycelium increased.After treated with fengycins for 12 and 48 h,the content of chitin increased by 41.32%and 46.02%,respectively,compared to the control group.The ergosterol content increased by 30.53%treated with 0.5 MIC fengycins compared to the control group.The biosynthesis of cell wall and membrane were the main target of fengycins against A.solani.3.Acetophenone is a key antifungal component of B.subtilis ZD01 volatile compunds.Through testing the biocontrol activity of volatile organic compounds(VOCs)produced by B.subtilis ZD01,VOCs significantly inhibited the lesion area expansion,as well as mycelial growth and spore germination of A.solani.The optimized headspace solid-phase microextraction gas chromatography-mass spectrometry(HS-SPME-GCMS)was used to analyze and identify VOCs.The 50/30 μm DVB/CAR/PDMS extraction head,with an extraction temperature of 50℃and an extraction time of 40 minutes was the optimal extraction condition.A total of 39 compounds with a matching degree of ≥ 800 were identified using two different polarity chromatographic columns.there were certain differences in the volatile components detected by HP-5 and FFAP chromatographic columns.The HP-5 chromatographic column detected 24 types organic compounds,with aromatic compounds accounting for the majority;The FFAP chromatographic column has the highest relative content of ketone organic compounds,with 10 unique compounds detected.Five compounds were detected in both two chromatographic columns.Acetophenone was detected with a relatively high content and significant antifungal effect,an EC50 concentration of 33.45 μL/L.4.B.subtilis ZD01 volatile substance acetophenone mainly regulated the membrane transport gene Hxt of A.solani,leading to ROS accumulation.Transcriptome sequence analysis was used to further explore its antifungal mechanism.The results showed that acetophenone mainly regulated the expression of key genes in the process of cell membrane transport protein and oxidative stress in A.solani,and the differential genes were mainly concentrated in the MFS(major facilitator superfamily)family.Hxt gene encoding the membrane hexose transporter protein was the most significantly differentially expressed after acetophenone treatment.Acetophenone caused complex dynamic changes in cell membrane proteins of A.solani,and the secondary structure of membrane proteins changing from βantiparallel to helix structure,and the accumulation of ROS in A.solani.To verify the function of Hxt gene,mutant strain construction was performed.mutants A Hxt was not sensitive to acetophenone,and this gene plays an important role in the pathogenicity process of A.solani.Using Alphafold technology,it was predicted that acetophenone could spontaneously bind to HXT,and the main types of interactions at the binding sites were hydrogen bonds and van der Waals forces.MET1,ASN134,MET150,PHE4 and TRP146 were the main amino acid residues involved in the interaction between acetophenone and HXT protein.In this study,a strain B.subtilis ZD01 with great biocontrol effect against A.solani was obtained.The main antifungal ingredients in antifungal lipopeptides and volatiles were identified as fengycins and acetophenone,respectively.The inhibitory mechanism of active component fengycins in supernatant was analyzed.This study revealed the regulatory effect of acetophenone,the main active component of volatile substances produced by B.subtilis ZD01,on MFS family hexose transporter Hxt gene in A.solani.The preliminary function of Hxt gene in A.solani was identified.In this study,the activity and mechanism of the main components of different types of secondary metabolites secreted by B.subtilis ZD01 against A.solani were analyzed in depth.It conducted the first study on the inhibitory mechanism of a specific volatile organic compound,acetophenone,against plant pathogenic fungi.The research provides a theoretical basis for the development of new biocontrol agents for the prevention and treatment of potato early blight. |
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