Combined Antioomycets Effect Of W-7 Antagonistic Strain Cell Wall Degrading Enzyme Induced By Phytophthora Infestans And Metalaxyl Mancozeb

Potato（Solanum tuberosum L.）is the fourth largest food crop in the world,and its planted area and output in China has been increasing year by year.Potato late blight caused by Phytophthora infestans（Mont.）de Bary is not only the primary disease in potato production,but also recognized as the world’s largest crop disease.The production mainly relies on disease-resistant varieties and chemical pesticides for prevention and control of disease.However,in recent years,the frequency of the mating type of P.infestans A2 has gradually increased,and the probability of sexual reproduction of the pathogen has increased.In addition,the excessive use of chemical pesticides has made P.infestans resistance increased,and the prevention and control of potato late blight has become more and more difficult.Therefore,timely monitoring and grasping of the dynamic changes and regularity of resistance of P.infestans to chemical pesticides（metalaxyl and its derivatives,etc.）are essential for formulating disease prevention strategies,especially the scientific and reasonable selection of pesticides.At the same time,the use of antagonistic bacteria to control potato late blight has attracted more and more researchers’attention,but its antioomyctes active substances need to be further clarified;in addition,the mixed use of antagonistic bacteria and pesticides to inhibit P.infestans is also rarely reported.The main research contents of this experiment are as follows:（1）Try to use potato tuber slices to monitor the feasibility of P.infestans resistance and establish a detection method;（2）It is clear that P.infestans induces W-7 strain to produce cellulose,at the same time whether this induction has an effect on the production or activity ofβ-1,3-glucanase,and to compare the effects of these two enzymes in inhibiting the growth of P.infestans mycelia;（3）Explore combined antiomycets effects of using the chemical pesticide（metalaxyl mancozeb）and Bacillus pumilus（Meyer and Gottheil.）W-7 on P.infestans and their disease-preventing effects on detached potato tissues.1.In order to monitor the metalaxyl resistance of P.infestans more timely and accurately,this experiment was conducted to test the possibility of potato tuber slices for detecting the metalaxyl resistance.After comparing the tuber slices floatage,filter paper adsorption,sponge adsorption,and direct daubing methods,we found that tuber slices could not float on metalaxyl solution surface,and both filter paper adsorption and sponge adsorption methods including control as sterile distilled water resulted in tuber slices rot easily.Only direct daubing metalaxyl solution on tuber slice surface under moisture culture could differentiate control and different treatments,and evaluate the resistance degree of P.infestans to metalaxyl,since there were significant differences in mycelia growth and tuber slice discoloration between control and different treatments at different metalaxyl concentrations.In addition,the appropriate concentrations of metalaxyl solution were optimized for this method,and they were between 5μg/m L and 80μg/m L,respectively.To utilize this method,we also proposed the criteria for resistance evaluation.The establishment of this method can provide a new selection for timely and accurately detecting metalaxyl resistance of P.infestans in different potato production areas in future.2.In order to understand deeply whether the bacterium W-7 inhibit the mycelia growth of P.infestans by producing cell wall degrading-enzymes,the dual culture method was used to detect the activity ofβ-1,3-glucanase and cellulase in cell-free agar block of the inhibition zone between W-7 and P.infestans colonies（LWK）,which were produced and secreted by W-7 strain after induction by P.infestans.Then the activity inhibitors of two enzymes were screened,and the anti-oomycetes effects of LWK on P.infestans mycelia growth were investigated after restraining activities of these enzymes in LWK by inhibitors.The results showed that bothβ-1,3-glucanase and cellulase were found in LWK,but onlyβ-1,3-glucanase in the cell-free agar block for culturing W-7 strain alone（DWK）.The activities of two enzymes in LWK reached the highest when dual culture of W-7 and P.infestans were conducted at 12 d,which were 22.682 U/m L and 4.785 U/m L,respectively.Of them,the activity ofβ-1,3-glucanase in LWK was significantly higher than that in DWK at 12 d（16.267U/m L）.After six substances were tested,it was found that SDS had relatively strong ability for inhibitingβ-1,3-glucanase activity and promoting cellulase activity,while Fe³⁺was exactly opposite to SDS,and Cu²⁺had the strongest capability for inhibiting activities of bothβ-1,3-glucanase and cellulase,simultaneously.Furthermore,the colony diameters of P.infestans after Cu²⁺treatment were the largest（45-57 mm）,followed by SDS treatment（36-47mm）,and that of Fe³⁺treatment were the smallest（31-44 mm）.There were significant differences in colony diameters of P.infestans between Fe³⁺treatment and control with normal enzyme activities,and that appeared between Fe³⁺and SDS treatment,also,while colony diameters of P.infestans after Cu²⁺treatment were significantly higher than that of both Fe³⁺and SDS treatment.Next,the effects of the agar block containingβ-1,3-glucanase and cellulase on the mycelium deformation and sporangia deformation and germination of P.infestans were tested.After treating them with different enzyme inhibitors,P.infestans mycelia treated with 10 mmol/L Fe³⁺solution were found that the distortion rate was 41.26%,the sporangia distortion rate was 29.26%,both of which were the highest,and the sporangia direct germination rate was the lowest（8.16%）.It indicated that W-7 strain could not only produce cellulase but produce moreβ-1,3-glucanase after induction by P.infestans.When the two enzymes exist at the same time,the suppression efficiency of LWK on P.infestans mycelia growth were significantly better than that of the two enzymes alone,in which anti-oomycetes effect ofβ-1,3-glucanase was remarkably stronger than that of cellulose and it could play a major role,while cellulase could exert some certain synergistic inhibitory effect when inhibiting P.infestans mycelia growth.3.In order to explore the inhibition potential of mix use of chemical pesticides and biocontrol bacterium on P.infestans,the mixture of B.pumilus W-7 strain and the metalaxyl mancozeb（MM）were cultured for 48h and spreading plate,the calculation colonies of W-7strain were conducted to determine the feasibility and optimum concentration of W-7 and MM compound.Dual culture method was used for detecting the effect of compound liquid（CL）on the shape and growth of P.infestans mycelia,mixture cultivation method for observing the effect of that on shape and direct germination of pathogen sporangia,and tuber slice method for evaluating the effect of that on disease prevention.The results showed that 15μg/m L MM had no effect on growth of W-7 strain.The inhibition rate（98.94%）of CL of MM and W-7strain at 1:2 volume on P.infestans mycelia growth and the mycelial deformation rate of CL（48.56%）was evident higher than sum of MM and W-7 treatment alone.The pathogen sporangia direct germination rate treated with CL（5.87%）was remarkable lower than that of W-7 strain and MM treatment alone,and sporangia deformation rate（53.88%）after treatment with CL was much similar to sum of MM and W-7 treatment alone.In addition,the disease prevention effect of CL（91.36%）on potato tuber slices was significantly higher than sum of MM and W-7 treatment alone.These findings indicate that mixture of W-7 strain and the metalaxyl mancozeb（MM）would greatly reduce the use of chemical pesticides for control potato late blight in future.In summary,this experiment clarified that potato tuber slices can be used to detect the resistance of P.infestans to metalaxyl,and a simple and effective method with reliable detection results was initially established,which is fast and accurate in production.It provides a new option to detect the resistance of P.infestans to metalaxyl and its derivatives on a large scale.At the same time,it is clear that B.pumilus W-7 can not only produce cellulase,but also produce moreβ-1,3-glucanase under the induction of P.infestans,and in which for inhibiting P.infestans,β-1,3-glucanase played a more important role;In addition,it was found that the compound of the antagonistic bacteria W-7 and metalaxyl mancozeb has a better inhibitory effect on P.infestans and has a better disease prevention effects in in vitro tissues,thses findings can provide a reference for reducing the use of pesticides while more efficiently preventing potato late blight.