Optimization And Analysis The Culture Conditions And The Key Proteins Of Trichoderma Longibrachiatum T6 Against Heterodera Avenae

Heterodera avenae is an important plant parasitic nematode,which mainly harms gramineae crops,and is distributed in the main wheat producing areas all over the world,and causes serious economic losses.At present,chemical control is still the main method to control cereal cyst nematode disease,but the chemicals has some disadvantages,such as large amount of chemicals use,polluting the environment and endangering the health of human and animal.Therefore,current research focus on the development of efficient and safe,environmentally and friendly biological nematicides.In this study,selected as test materials,which had good effects on heterodera avenae,medium screening and optimizing culture conditions of Trichoderma longibrachiatum T6,the stability of fermentation broth was tested,the protein mass spectrometry was detected,and nematicides protein was isolated.The aim of this study was to reveal the toxicity mechanism of Trichoderma longibrachiatum T6 to Heterodera avenae and provide theoretical basis for biological control of Heterodera avenae.The main results are as follows:1.Screening for the optimum medium for nematicides of T.longibrachiatum T6 as casein medium.Though single factor test,obtaining the optimum condition of nematicides include pH=7,28?,fermentation time is 7 days,inoculation volume is 1-2 mL,liquid medium volume is 50-70mL.According to the results,temperature,pH and fermentation time were the most significant factors,which affect the activity of fermentation broth,so using the response surface methodology to optimize three factors.The result show that pH=6.64,26.52?,6.8d as the optimum fermentation conditions of strains T6 under this condition,the actual lethality rate was86.10%,which was close to the predicted value,which was close to the predicted value.2.The stability of acid-base,temperature,metal ions,redox and storage time of T.longibrachiatum T6 fermentation broth were tested,and the stability of fermentation broth was evaluated comprehensively.The results showed that the fermentation broth had a high lethal effect to nematodes in the acid-base pH range of 4 to 10.The mortality rate of nematodes at 72h was more than 80%,and there was no significant difference at the pH range of 4 to 10.The temperature stability test showed that after 30-60?water bath treatment for 30 min,the nematicides activity of fermentation broth had no significant difference under different temperature.When the temperature exceeded 70?,the nematicides activity of fermentation broth decreased.Metal ion stability test showed that Zn²?and K?impoved the effect of nematicides activity of fermentation broth,Cu²?could decreased the effect of nematicides activity of fermentation broth,while Mg?,Fe³?,Mn²?and Na?had no significant effect.Oxidation-reduction stability test showed that concentration of oxidant and reductant was lower than 0.08 mol?L^-1 and 0.06 mol?L^-1 respesentively.There was no significant effect on the activity of the fermentation broth,and the stability test during storage showed that there was no significant effect on the nematicides activity of the fermentation broth when it was stored less than 60 days at room temperature.3.The protein of T.longibrachiatum T6 was detected by mass spectrometry,and the spectrum 39537 spectra and four Trichoderma proteins was obtained,after matching the protein database of Trichoderma spp and prediction of physicochemical properties and structures of proteins by amino acid sequences.The GO function of the matching protein was annotated to participate in three molecular functions:transporter activity,catalytic activity and enzyme binding activity.The analysis of cell component showed that there were matches in cell,cell membrane,organelle and so on.Protein exists and participates in five biological processes,including cellular process,localization,response to stimulus.COG annotation functions include posttranslational modification,protein turnover,chaperones,Energy production and conversion,Amino acid transport and metabolism.4.The ability of T.longibrachiatum T6 to produce extracellular proteins was qualitatively and quantitatively analyzed by transparent circle method and spectrophotometry,which indicated that T.longibrachiatum T6 could produce extracellular proteins.Neutral salting-out method and column chromatography were used to isolate the nematicides activity proteins from T.longibrachiatum T6 fermentation broth.The results showed that three groups of elution peaks were obtained.Among them,elution peaks A and B had strong nematicides activity.When the concentration of them were 12.48?g.mL^-1 and 9.20?g.mL^-1 respectively,the mortality rate of nematode treated for 72 hours was 52.05%and 43.89%,while the low nematicides activity of elution peak C,which was only 22.42%.