Identification And Biocontrol Of Two Antagonistic Bacteria Against Sclerotinia Sclerotiorum In Oliseed Rape

Oilseed rape is one of the main oil crops in China.Sclerotinia stem rot caused by Sclerotinia sclerotiorum（Lib.）de Bary is one of the most important diseases in rape production.At present,the main measures to control the disease are chemical control,combined with agricultural control.However,due to the adverse effects of chemical agents on the environment and biological health,agricultural control measures are time-consuming and laborious.People gradually turn their attention to environmental protection and long-lasting control measures.The use of antagonistic microorganisms for biological control has become a hot research field of scholars at home and abroad.Among them,the use of soil biological control bacteria and sclerotial endophytes for the control of the disease of rape is more important and outstanding.In this paper,the screening,identification and biocontrol of antagonistic bacteria against Sclerotinia sclerotiorum were studied.The main results are as follows:1.Isolation,screening,antimicrobial activity and identification of biocontrol bacteria against Sclerotinia sclerotiorum in oilseed rapeFive strains of biocontrol bacteria were isolated and screened from the soil samples of Hefei and Wuhu,Anhui Province by the method of soil dilution coating.By plate confrontation test,strain TR-17 had the best bacteriostatic activity;six strains of endophytic bacteria with antagonistic effect were isolated and screened from the sclerotia of Sclerotinia sclerotiorum.Among them,strain NS-19 had good genetic stability and strong bacteriostatic effect,and the inhibition rates of the two strains were 74.45%and 69.09%respectively.According to the morphological observation,16S r DNA sequence analysis and physiological and biochemical test results,the strain tr-17 was identified as Bacillus subtilis,and the strain NS-19 was B.amyloliquefaciens.2.Effects of antagonistic bacteria on the growth of Sclerotinia sclerotiorumUnder the light microscope,the growth of Sclerotinia sclerotiorum mycelium cultured in confrontation with the antagonistic strain was observed.It was found that the Sclerotinia sclerotiorum mycelium affected by strain TR-17 was thin and broken,and its branches were not obvious;the top of the mycelium affected by strain NS-19was expanded,the mycelium was thin and its morphology was distorted.The results showed that the inhibition spectrum of TR-17 and NS-19 were narrow,and the inhibition rate of TR-17 and NS-19 was less than 50%.However,TR-17 had 72.42%,93.79%and 55.78%inhibition rates on Pyricularia oryzae Cav,Gaeumannomyces graminis,and Botrytis cinerea.NS-19 had 92.41%and 51.47%inhibition rates on Gaeumannomyces graminis and B.cinerea.Clearly,both of the above two biocontrol strains had more than 92%of inhibition against G.graminis.3.The optimum growth conditions of antagonistic bacteria,the determination of antibacterial activity of fermentation broth and the analysis of toleranceThe optimum culture conditions of strain TR-17 were:initial p H 6,culture at28℃for 48 h.The most suitable carbon source was glucose and the most suitable nitrogen source was sodium nitrate.The antibacterial activity of TR-17 aseptic fermentation broth was strong,and the antibacterial effect increases with the increase of aseptic fermentation broth content,but decreases with the increase of culture days.With 4.634%of EC₅₀,TR-17 aseptic fermentation broth was susceptible to the influence of temperature,p H and UV irradiation.The optimal growth conditions of strain NS-19 were:initial p H 8,culture at 28℃for 48 h.The optimum carbon and nitrogen sources were xylose and glycine.The inhibition effect of NS-19,with1.383%of EC₅₀,was better than that of TR-17,and the tolerance of NS-19 was better than that of TR-17.4.Preliminary analysis of antifungal components of free fermentation broth of antagonistic bacteriaThe antibacterial crude protein was precipitated by ammonium sulfate of different saturation,and the antibacterial activity of the corresponding crude protein was measured.The experimental design showed that the addition amount of crude protein was 10%.When the saturation of ammonium sulfate was 90%,the inhibitory activity of the antibacterial protein extracted from TR-17 was the highest,which was70.75%;when the saturation of ammonium sulfate was 70%The bacteriostatic rate of the crude protein extracted by NS-19 was 44.38%.By acid precipitation and methanol extraction of lipopeptide antibacterial substances from aseptic fermentation broth,the results showed that the antibacterial activities of two antagonistic lipopeptide roughs were higher than that of their protein roughs,and with the increase of the concentration of lipopeptide roughs,the antibacterial activities increased accordingly.When the content of TR-17 lipopeptide roughs was 2.5%,the inhibition rate could reach 81.88%.When the crude content of NS-19 was 4.5%,the inhibition rate was90.42%.Therefore,it is preliminarily determined that the main antibacterial component of the two antagonistic bacteria free fermentation broth is lipopeptide.The gene primers for the synthesis of lipopeptide were designed.The whole genome of strain NS-19 was used as template to amplify the gene fragments of lipopeptide antibiotics such as iturin,fengycin and surfacin.Combined with LC-MS,the composition of lipopeptide of strain NS-19 was determined.The two-stage mass spectrometry matched many amino acids and organic acids.These amino acids are the main components for the synthesis of lipopeptide antibiotics.It is concluded that the single bacteriostatic substance of lipopeptide crude extracted by NS-19 may include iturin,fengycin,surfacin,etc.The antibacterial spectrum of the NS-19 lipopeptides was measured by agar pore diffusion method,and the results showed that among the 10 pathogens tested,there were 6 pathogenic bacteria with a width of 0.70 cm or more,respectively:R.cerealis,G.graminis,F.oxysporum,A.alternata,S.sclerotiorum,and V.mali.5.Effects of antagonistic volatile substances on S.sclerotiorum growth and sclerotial germinationThe volatiles produced by the two antagonistic strains had a certain effect on the growth of S.sclerotiorum,According to the results of double dish inverted clasp culture,the inhibition rate of volatile substances on S.sclerotiorum mycelium was89.38%when TR-17 was diluted 100 times and coated.The inhibition rate of volatile substances on S.sclerotiorum mycelium was 64.06%when NS-19 was coated with the same concentration.The above volatile substances can delay the germination of sclerotia,but can not completely inhibit the germination of sclerotia.6.In vitro leaf test and pot experimentAfter inoculation with strain TR-17 and strain NS-19 aseptic fermentation broth,the inoculated S.sclerotiorum did not develop on the first day.The average diameter of lesion spots was 1.04 cm and 0.78 cm on the second day,and the average spot diameter of the control group reached 1.69 cm at the same time.The leaves treated with NS-19 lipopeptide showed no lesion in the first two days.Using the above fermentation broth and lipopeptide boldface,spray treatment of stem and base of potted rape leaves and potted plants respectively,and the control effect was remarkable.The greenhouse was placed at 25℃,and the leaves of three groups had no disease.The average spot size of the control group was 2.23 cm on the second day.There was slight disease at the base of stem treated by TR-17 fermentation liquid（one of the three rape plants was infected）,the epidermis at the base of stem was rotted in a small area,and there was no disease in the other two treatment groups.In the control group,the epidermis at the base of stem was rotted in a large area,the plant collapsed and the leaves were withered.