Anti-oomycetes Action Of Cellulase Produced In W-7 Straian Induced By Phytophthora Infestans

Potato?Solanum tuberosum?is one of the world's staple foods,it has the characteristics of strong adaptability,high yield,and rich nutritional value.The planting area,yield and sales volume of potatoes in China are constantly increasing.However,the production of potatoes often suffers from many pathogens.Among them,the potato late blight caused by Phytophthora infestans was recognized as the number one largest crop disease in the world and has been the concern of many international researchers'focus.At present,the yield and quality safety of potatoes had become a problem to be solved.Therefore,it is urgent to explore new control methods with the characteristics of safety,high efficiency,no pollution,and prevention of pathogen resistance.In recent years,our laboratory has screened a number of strains that could significantly inhibit the growth of P.infestans mycelium and spore germination.Among them,an antagonistic W-7 strain is particularly prominent.On the one hand,its bacterial solution has good thermal stability,acid and alkali resistance,and certain UV stability;on the other hand,the strain has the very significant inhibitory effect on P.infestans,and the inhibition rate can reach 94.44%.And it showed the very good disease prevention effect on potato tubers,the prevention effect could reach 73.40%;what's more,the strain was isolated from potato leaves,compared with the antagonistic bacteria isolated from other places,it has very prominent advantages in colonization on potato plants and has great potential for biocontrol.This article intends to explore the antibacterial active substances produced by bacteria W-7 and its stability and can provide experimental basis for clarifying its mechanism of inhibiting P.infestans.In previous research in our laboratory,it has been found that the W-7 strain could cause many morphological changes in the mycelia of P.infestans,especially the mycelium often appears rough,uneven,locally enlarged,and accumulation of contents or overflow.The morphology of mycelium is maintained by the cell wall.The morphological distortion of the mycelium indicates that the skeleton structure of the cell wall of the mycelium has been changed,which means that the main structural substances of the cell may have been changed or even been destroyed.P.infestans belongs to oomycetes,and cellulose is one of the main structural substances constituting for its cell wall.Based on this,we speculate that the W-7strain may change the cellulose in the cell wall of P.infestans mycelium by producing cellulase,which may cause mycelial aberration and inhibit mycelial growth.P.infestans does not produce cellulase by itself.Therefore,the main research contents of this trial include:?1?To investigate whether the cellulase can be produced by Bacillus pumilus W-7 when cultured against P.infestans by dual culture method and Congo red staining method.?2?Using the DNS method to clear that the effect of bacteria W-7 and P.infestans dual culture at different times,as well as the bacteria W-7 after culturing separately under different conditions of temperature,pH,nitrogen source and inoculation amount co-cultured with P.infestans on cellulase-producing activity in W-7 strain and the inhibitory effect of cellulase-containing agar blocks on the growth of P.infestans and to select the best culture conditions;?3?To investigate the stability of cellulase produced by P.infestans-induced bacteria W-7 under different conditions,including different temperatures,pH,salt concentration,metal ion,substrate concentration,organic substances by measuring the cellulase activity,and to understand the enzymatic properties of cellulase produced by W-7.The main research results obtained in this experiment are as follows:1.It was clarified that the W-7 strain did not produce cellulase by itself,but cellulase was produced during the dual culture with P.infestans,indicating that P.infestans stimulated or induced W-7 strain to produce cellulase.2.The effect of cellulase-containing agar blocks treated with extremely acidic or extremely alkaline buffers?pH 3.5 and pH 10?on the mycelial growth of P.infestans was researched.According to the transparent dissolution circle produced by the free cell agar block,compared with the control group,it was evident that the acid or alkali could inhibit the cellulase activity at a certain extent.The inhibition rates of the two treatments on the growth of P.infestans were 29.78%and 30.11%respectively,the control groups was 71.52%and 0%.3.The effect of free cell agar block containing cellulase treated with extremely acidic?pH 3.5?or alkaline buffers?pH 10?on mycelium teratogenicity of P.infestans was microscopic observed.The results showed that after the ellulase-containing agar blocks were treated with buffers?pH 3.5 or pH 10?and co-cultured with P.infestans on Rye for 7 days,the mycelium at the edge of the colony of P.infestans was deformed.The teratogenic rate of mycelia were 12.61%and 14.21%,the control group was 38.43%.4.The W-7 strain was optimized for separate culture conditions in advance,and then the cellulase production of W-7 and its teratogenic rate on P.infestans mycelium were determined under dual culture of W-7 with P.infestans.The conditions for separate culture of W-7 were 25?,the initial pH 7 and?NH₄?₂SO₄ as the nitrogen source,and the W-7 was co-cultured with against P.infestans that all had the highest cellulase activity?4.53U/mL,5.28 U/mL and 5.89 U/mL?and the highest mycelium teratogenic rate?31.82%,34.49%and 37.87%?.After the bacterial solution of W-7 natural mixed colonies was diluted with sterile water,the highest cellulase activity was 5.91 U/mL and the highest mycelium teratogenic rate was 34.78%.5.The optimal reaction conditions of cellulase and the effects of different conditions on cellulase stability were investigated.?1?The cellulase produced by W-7 strain was not heat-resistant,and its thermal stability was poor,and the optimum reaction temperature of the enzyme was 50?.?2?The cellulase produced by bacteria W-7 was a weak acid enzyme and could maintain good stability in a weak acid environment.?3?Ca²⁺,K⁺,Fe²⁺,and Mn²⁺with the concentration of 1mmol could increase the activity of the crude enzyme,while treatment with the concentration of 10mmol of these metal ions could inhibit cellulase activity;?4?The concentration of 1%and 10%methanol and ethanol could promote the enzyme activity of cellulase,while the concentration of 15%and 30%methanol and ethanol would weaken the enzyme activity,both isopropanol and ethyl acetate could inhibit the enzyme activity of cellulase;in addition,the cellulase produced by the W-7 strain has good tolerance to salt.