Research On The Control Of Strawberry Verticillium Wilt By Compost Tea And Its Action Mechanism

Verticillium wilt of strawberry,caused by Verticillium dahliae Kleb.,is a devastating vascular wilt disease,which has become the bottleneck restricting the sustainable development of strawberry industry.Due to the lack of resistant strawberry cultivars,management of Verticillium wilt is achieved primarily through pre-plant soil fumigation or crop rotation,but these approaches have some drawbacks and limitations.Therefore,there remains an extremely urgent need to develop safer and highly efficient strategies to control strawberry Verticillium wilt.Previous studies have shown that compost teas derived from agricultural solid wastes have inhibitory effects on numerous phytopathogens,suggesting that compost teas can be exploited as valid alternatives to chemical fungicides for use in crop protection.However,the efficacy of compost teas varied considerably depending on the compost type,the preparation and application methods of the tea.Taking into account the complex physicochemical and microbial composition of compost teas,it is often difficult to determine the exact mode of action.In view of all the above,the main objectives of this study were to optimize the parameters for the extraction of spent mushroom substrate（SMS）compost tea and maize straw（MS）compost tea,assess the biocontrol efficacy of compost teas to suppress Verticillium wilt of strawberry and identify the dominant mechanisms involved in the preventing and controlling of this disease.This research can provide a theoretical basis and technical support for the production and practical application of compost teas.In addition,functionally active microbes isolated from the compost teas were screened for their antagonistic activity against Verticillium wilt pathogen,and the taxonomic status,biocontrol efficacy and antimicrobial metabolites of antagonist were systematically studied.This study will lay the foundation for future development of microbial pesticides to protect strawberry plants against Verticillium wilt disease.1.Compost teas were prepared from SMS compost and MS compost under continuous aeration or non-aeration condition,and factors affecting the suppressiveness of compost teas against V.dahliae were optimized.The differences in microbial community structure and diversity in composts and compost teas were uncovered using Illumina Nova Seq sequencing.The optimized conditions for extraction of aerated SMS compost tea were determined to be compost to water ratio of 1:4（w/v）,extraction temperature of 25°C,extraction time of 3 days and minimum dissolved oxygen concentration of 6.0 mg/L.Optimum conditions for non-aerated SMS compost tea extraction were compost to water ratio of 1:4（w/v）,extraction temperature of 30°C and extraction time of 6 days.The optimized conditions for extraction of aerated MS compost tea were determined to be compost to water ratio of 1:6（w/v）,extraction temperature of 30°C,extraction time of 2days and minimum dissolved oxygen concentration of 6.0 mg/L.Optimum conditions for non-aerated MS compost tea extraction were compost to water ratio of 1:6（w/v）,extraction temperature of 35°C and extraction time of 6days.High-throughput sequencing results revealed that the bacterial richness in compost teas was higher than in composts.Furthermore,alpha-diversity analysis of compost tea samples showed that compost teas extracted under continuous aeration had higher bacterial and fungal community diversity than non-aerated compost teas.The structure of microbial communities varied greatly among compost teas extracted under different conditions.However,the dominant bacterial phyla shared by all compost tea samples were Firmicutes,Actinobacteria,Bacteroidetes and Proteobacteria.The predominant bacterial genera in aerated SMS compost tea were Arachidicoccus,Taibaiella,Olivibacter and Pedobacter,while aerated MS compost tea was dominated by members of the genus Salinispora and Bacillus.In addition,Ascomycota was the most dominant fungal phylum in all compost tea samples.By comparing with SMS compost,the relative abundance of Hapsidospora,Thermomyces,Mycothermus and Crassicarpon in aerated SMS compost tea remarkably increased during the extraction process.Aerated MS compost tea had higher relative abundance of Penicillium,Trichoderma,Sterigmatomyces and Peniophora than MS compost.Comparing the culturable microbial population,microbial diversity and inhibitory effect of compost teas extracted under different conditions,it was concluded that aerated compost teas are superior to non-aerated compost teas.2.In vitro and in vivo trials were conducted to assess the control efficacy of the teas prepared from SMS compost and MS compost against Verticillium wilt of strawberry and the mechanisms involved in suppression of the phytopathogen.The SMS compost tea used in the present study inhibited the mycelial growth and conidial germination of V.dahliae by92.43%and 82.94%,while MS compost tea showed inhibition rate of90.60%and 78.24%,respectively.However,sterilization of compost teas by autoclaving or microfiltration diminished the inhibitory effect,suggesting that microflora in the compost teas play a pivotal role in the prevention of Verticillium wilt.Greenhouse trials indicated that preventive application of SMS compost tea and MS compost tea on strawberry plants exhibited control efficacy of 42.23%and 41.85%against Verticillium wilt,respectively.Moreover,both SMS compost tea and MS compost tea treatments could induce resistance in the primed state,which led to enhanced production of defense-related enzymes in strawberry plants upon pathogen attack.The results demonstrated that direct inhibition of V.dahliae by microorganisms and induction of plant defense response are main mechanisms involved in compost tea-based disease suppression.3.To obtain potential antagonists that can be integrated into disease management strategies,189 isolates of bacteria were collected from compost teas and screened for their antagonistic activities against V.dahliae.Among these isolates,three bacterial strains possessed high antifungal activity against the target pathogen,showing inhibition zones greater than 8 mm and inhibition rates greater than 65%.One of these bacterial strains,designated as CT32,was found to have the highest antagonistic activity,showing an inhibition rate of mycelial growth of 74.86%.The strain CT32 was identified as Bacillus velezensis based on 16S r RNA gene sequence similarity,morphological characteristics,Biolog GEN III and API 50 CH analyses.Greenhouse trials indicated that preventive application of strain CT32 on strawberry plants exhibited control efficacy of 58.82%against Verticillium wilt.In addition,preventive application of the bacterium CT32 suspension increased plant height,stem diameter,aerial fresh weight and dry weight,root fresh weight and dry weight of infected strawberry plants by 47.48%,51.35%,63.75%,59.16%,66.81%and 75.14%,respectively,as compared to the control.These results indicate that B.velezensis CT32 has the potential to be developed into a biocontrol agent for management of strawberry Verticillium wilt.4.The volatile organic compounds（VOCs）produced by B.velezensis CT32 were qualitatively analysed and tested for their antifungal activities.A method based on headspace solid-phase microextraction/gas chromatography-mass spectrometry（HS-SPME-GC-MS）was developed to analyse the VOCs emitted by strain CT32.Optimal extraction of bacterial volatiles using 50/30μm DVB/CAR/PDMS fibre was 53 min at 74°C with 27 min equilibrium time.The chromatographic separation condition on an HP-5MS capillary column was initial 40°C with a 2 min hold,ramped to 110°C at 4°C/min,ramped to 130°C at 2°C/min（held for 2 min）,ramped to 150°C at 2°C/min（held for 2 min）,ramped to 200°C at 3°C/min,ramped to 300°C at30°C/min（held for 5 min）.A total of 50 volatile compounds were isolated,of which 31 were identified,accounting for 90.85%of the total volatile components.Six of these compounds have not previously been detected in bacteria or fungi:2,2’,5,5’-tetramethyl-1,1’-biphenyl,2-methylpentadecane,dodecanenitrile,2,4-dimethyl-6-tert-butylphenol,（Z）-5-undecene and decyl formate.The results obtained in the in vitro assays confirmed the antifungal activity of strain CT32 towards eight plant pathogens and indicated the pivotal role of VOC production in antagonist–pathogen interactions.Pure compounds were tested for their inhibitory effect on the mycelial growth of V.dahliae and Fusarium oxysporum f.sp.fragariae.Decanal,benzothiazole,3-undecanone,2-undecanone,2-undecanol,2,4-dimethyl-6-tert-butylphenol and undecanal showed high antifungal activity,with benzothiazole and2,4-dimethyl-6-tert-butylphenol being the most potent compounds.5.The antifungal spectrum of B.velezensis CT32 was determined,and the genes involved in biosynthesis of antimicrobial compounds were detected.One-factor test and Plackett-Burman design were used to screen key variables affecting the biosynthesis of lipopeptides by strain CT32.Response surface methodology was employed for the optimization of medium constituents and fermentation conditions for lipopeptides production by the bacterium.Results obtained from in vitro assays showed that strain CT32possessed broad-spectrum antifungal activity against eight phytopathogenic fungi.The genes encoding antimicrobial compounds,including srf AB,itu C,fen D,bmy B,mln A,bae N,bac B,dfn A and dhb F,were successfully amplified from the genome of this bacterium.The results indicated that strain CT32 has the capacity to synthesize surfactin,iturin,fengycin,bacillomycin,macrolactin,bacillaene,difficidin,bacilysin and bacillibactin.Among the 14variables investigated,concentration of NH₄NO₃,concentration of Mg SO₄,incubation temperature and medium volume were identified as key variables which significantly influenced lipopeptides yield.The optimized medium constituents for maximum yield were found to be 40.0 g/L of glucose,4.88g/L of NH₄NO₃,0.47 g/L of Mg SO₄·7H₂O,0.3 g/L of KCl,1.8 g/L of KH₂PO₄,0.15 mg/L of Fe SO₄·7H₂O,5.0 mg/L of Mn SO₄·H₂O,0.16 mg/L of Cu SO₄·5H₂O and p H value of 6.0.Optimum fermentation parameters were incubation temperature of 31°C,fermentation time of 48 hours,inoculum size of 6%（v/v）,medium volume of 64 m L per flask and rotation speed of200 rpm.The lipopeptides yield under the optimized conditions was 1.57 g/L,which was increased by 106.58%compared to an earlier value of 0.76 g/L before the optimization.