Fermentation Of Biocontrol Agents Of Valsa Sordida And Study On Its Resistance Mechanism

As the state actively promotes the idea of the ecological environment protection,biological control technology became more and more valued.The method for inhibiting a microorganism or a class microorganism by one of another species can maximize the protection of the environment from pollution.With the aim to use bio-control methods to control poplar canker disease caused by Valsa sordida,which causes poor growth and even result in poplar trees death,an antagonistic Streptomyces strain which has significantly inhibition effect on Valsa sordida was isolated from soils in Jilin Province of China.Systematical studies were made of the antagonistic Streptomyces strain in such aspects as classification and identification,optimization of fermentation conditions and action mechanisms of inhibiting pathogens and controlling disease.It is of great practical significance in the development of a new-type high-efficient and non-toxic biological agents and provided new ideas and theoretical reference for taking bio-control methods to control poplar canker disease.The results are as follows:1.Optimization of fermentation conditions for biocontrol Streptomyces: The results of the screening of fermentation medium component: Biocontrol Streptomyces can use much of the kinds of carbon sources,nitrogen source,the best carbon source is the soluble starch,the best organic nitrogen source is peanut powder,inorganic nitrogen source is ammonium sulfate.The experimental results of orthogonal design of fermentation medium:the result of which was expressed in the following formula:70g soluble starch,25 g peanut powder,2g yester powder,3g NH4NO4,2.7g CaCO3,2.7g NaCl,1000 ml water,at pH7.2.The optimum conditions for fermentation in shake-flask were defined as follows: fermentation temperature,28?,initial pH at 7.0;fermentation time,5 days;five-day old strains cultivated on medium for seed broth preparation;the volume of broth in a 250 ml flask,40 mland inoculation amount,4.5%.Determination of the physical and chemical properties of the fermented broths showed that strain F57 were highly resistant to temperature,has antibacterial activity at the temperature of 28¹21,pH tolerance determination showed that the the antibacterial activity of pH 4¹4 was better,and strain 57 is all stable in stabilities against its storage tolerance and subculture,worth development and application.2.The result of study on antibacterial mechanism of antagonistic bacteria:The fermentation broth of strain F57 has obvious inhibition effect on mycelial growth.It can lead to electrolyte leakage of pathogenic bacteria,MDA content in mycelia increased.These results proved the action site of strain F57 is on the cell membrane.The result of the study of respiratory enzymes of pathogenic bacteria: The fermentation broth can inhibit the activity of HK and PK in glycolysis pathway,and promote the activity of PFK,thus affecting glycolysis.The inhibition of SDH activity in the TCA cycle prevented the TCA cycle from proceeding normally.Promoted the activity of Ca²⁺ and Mg²⁺-ATP,and make the energy conversion of cells abnormal.It also inhibited the activity of GSH-Px,resulting in the failure to remove large amounts of reactive oxygen species and damaged the cell membrane in time.Ultimately,it affected the metabolic system of pathogens and played a role in killing and inhibiting pathogens.3.The result of study on the induced resistance of host plants: The content of proline,flavonoid total phenol,soluble protein were increased in poplar after treatment with only fermentation liquid,and the energy of PAL increased.Those showed that the fermentation liquid enhanced poplar disease resistance.In this paper,we studied the expression of SA and JA signal transduction pathway related genes in poplar for the first time.The result showed that the PR-1 gene expression of poplar was up-regulated,and the PAL gene was up-regulated at the late growth stage,and the expression of COI1-1 and COI1-2 genes was slightly up-regulated after treatment with fermentation liquid,The fermentation liquid opened up SA and JA signaling pathways to induce poplar resistance.All in all,The optimization of the fermentation medium and conditions for fermentation in shake-flask was carried out.And the study on the mechanism of disease resistance showed that the fermentation broth of biocontrol bacteria can resist the disease by enhancing the resistance of poplar,it can also directly kill and inhibit valsa sordida of poplar.