Preliminary Study On The Interaction Between Phytophthora Infestans And Antagonistic Sy11

Since 2015 our country put forward the strategy of potato as staple food,China's potato planting area and output have continued to expand,currently ranking first in the world,and China has become the largest country in potato production and consumption.However,the potato late blight caused by Phytophthora infestans is regarded as the world's largest crop disease,and causes huge losses to the potato industry every year,and seriously restricts the development of the potato industry.At present,spraying chemical pesticides or breeding disease-resistant varieties are mainly used to prevent late blight in potatoes in agricultural production.As the long-term large-scale use of chemical pesticides P.infestans becomes more resistant than before to pesticides;meanwhile,the excessive use of chemical pesticides has already caused huge pollution of nature environment and the living environment of human beings.In addition,cultivating disease-resistant varieties is high cost and time-consuming,the speed of breeding disease-resistant varieties is far less than the mutation speed of P.infestans.Finding safer and more effective control measures has become the key to the development of the potato industry.Biological control has great potential in preventing and controlling agricultural diseases because of its advantages of high efficiency and environmental protection.The use of antagonistic bacteria and its metabolites to control potato late blight has become a hot research topic.At present,the research on P.infestans mainly focuses on the screening,identification,evaluation of bacteriostatic activity,isolation and purification of bacteriostatic substances,detection of disease prevention effects of detached tissues and pot plants,etc,but the physiological and biochemical mechanisms of P.infestans inhibited by antagonists and the induction phenomenon between antagonistic bacteria and P.infestans are less studied.In-depth research on the mechanism of physiological and biochemical changes in pathogens after being inhibited by antagonistic bacteria and the correlation between antagonistic bacteria and P.infestans will not only help to reveal the interaction mechanism between antagonistic bacteria and pathogens,but to efficiently use antagonistic bacteria for disease control.In our previous research,we screened an actinomycete Sy11?identified as Streptomyces miceroflavus?from tomato field soil,its living body and culture liquid containing cells cultivated certain conditions have the strong inhibitory effect on the growth of P.infestans mycelium and sporangium germination,however,its free cell fermentation liquid has no effect on P.infestans.Further research revealed that there is an induction phenomenon between Sy11 strain and P.infestans,and only when P.infestans exists,will Sy11 strains produce anti-oomycetes substances to inhibit the growth of P.infestans.Based on this,it is speculated that the P.infestans could produce some signal molecules when it is co-cultured with Sy11 strain,Sy11 strain produce substances that inhibit P.infestans growth after stimulated or induced by these signal molecules.However,the specific action and mechanism of this induction phenomenon between P.infestans and Sy11 need to be further verified and studied.This research is based on the above results to investigate following problems:?1?microscopic observation of the morphology of mycelia and sporangia of P.infestans after inhibited by Sy11 strain,and detection of soluble sugars,proteins,MDA,defensive enzymes,and related energy metabolism enzymes in the mycelium of inhibited.?2?analysis of the induction way between Sy11 strain and P.infestans,especially the situation for P.infestans to generate signal molecules;?3?preliminary isolation and extraction of signal molecules produced by P.infestans,and reveal the relationship between the production site and concentration of signal molecules and the induction effect;?4?The preliminary study of the physiological and biochemical properties of the crude extracts of signal molecules and the chemical identification of their main chemical components to provide a theoretical basis for clarifying the material structure of signal molecules and revealing the specific induction mechanism.The research results obtained in this study are as follows:1.Both the Sy11 living strain and the culture liquid containing cells cultivated certain conditions had a strong inhibitory effect on the mycelia growth of P.infestans,and the inhibitory rate reached more than 85%.The anti-oomycetes substances were produced by the Sy11 strain during dual culture of it with P.infestans and were easily soluble in ethyl acetate,and also had a relative strong inhibitory effect on the growth of P.infestans mycelium,inhibition rate reached 55.67%.Statistical analysis showed that after treatment with Sy11anti-oomycetes substance,the zoospore release rate of P.infestans was only 19.53%,and the direct germination rate of sporangia was only 13.6%,which were significantly different from the control group?37.03%and 35.46%,respectively??P<0.05?.The mycelium of P.infestans inhibited by Sy11 had obvious distortions,mainly manifested by rough surface,excessive branching,uneven distribution of contents or spills,right-angled bends,etc,as a result,causing a great deal of damage to the mycelia especially for cell wall;sporangia had no change in shape,but the contents distribution changed significantly and formed a"big eye"structure,after long-term inhibition by Sy11.2.After being inhibited by Sy11 strain,the soluble sugar and protein content in P.infestans mycelium were reduced,while the content of malondialdehyde were increased significantly,compared with control treatment.In the detection of defensive enzymes in inhibited mycelium,it was found that the levels of superoxide dismutase?SOD?,catalase?CAT?,and peroxidase?POD?were significantly increased.The enzyme contents of hexokinase?HK?,succinate dehydrogenase?SDH?and adenosine triphosphate?ATP?,which are related to glycolysis,TCA cycle and energy metabolism,were significantly reduced,in comparison with control.And among them,ATP enzyme content was the the most decrease,which was reduced by 88.99%lower than that in control group.3.It was found that the signal molecules with induction role were produced by P.infestans when both it was cultured alone or co-cultured with Sy11.The signal molecules could spread through the medium while P.infestans grew,as Sy11 strain sensed these signal molecules,it produced anti-oomycetes substances to inhibit the growth of P.infestans.By analyzing the signal molecules produced by P.infestans,it was found that the signal molecules were not belong to protein or polysaccharides,they might belong to lipids or at least mainly lipids,and easily soluble in ethyl acetate.4.The signal molecule substances of P.infestans were separated and extracted with different methods and different organic solvents.The results showed that the molecular weight of the effective component of the signal molecule substances was between 1 000¹4000 D.After precipitation and extraction with different reagents,the strongest inhibition effect on P.infestans growth was found to be the signal molecule substances with absolute ethanol and direct extraction with ethyl acetate treatment from P.infestans free cell fermentation liquid.In addition,the signal molecule substances were red,and yellow brown oily osubstances after concentration and recrystallization.5.The crude signal extracts of P.infestans were found to be sensitive to high temperatures?90??and extreme acid and base?6>pH,pH>8?,but not sensitive to ultraviolet and protease treatments.In the presence of Cu²⁺or Zn²⁺ions,their activity were significantly reduced,which might be due to the formation of chelate between the signal molecule and Cu²⁺or Zn²⁺.The signal molecule extracts still could induce Sy11 strain even at a lower concentration,after it was diluted by 10⁵ times,the inhibition rate reached up 71.09%.6.The signal molecules of P.infestans existed in both intracellular and extracellular space.To analyze different development stage of P.infestans,it was found that the signal molecules extracted from all mycelium,sporangia and zoospores had a certain induction effect on anti-oomycetes action of Sy11 strain,and the inhibition rates after induction treatement reached more than 70%.Through chemical identification,the main effective components in crude extract of P.infestans signal molecules were preliminarily considered as the compound which contained the volatile oils,anthrones and glycosides,organic acids and amino acids.