A Study On Isolation Of Biocontrol Bacteria Y13 From Rehmannia Glutinosa And Its Control Of Rehmannia Glutinosa Leaf Ring Rot

Rehmannia glutinosa is a traditional Chinese herb used as a tuber in China.Medical research has shown that R.glutinosa has good therapeutic effects on cardiovascular diseases and central nervous system diseases,as well as pharmacological effects such as regulating blood sugar and blood lipids,anti-tumor,and enhancing immunity.It is mainly cultivated artificially,and is best produced in Jiaozuo,Henan Province（Huaiqing Province）,and is also cultivated in Shandong,Shanxi and Shaanxi.In recent years,with the increasing area of artificial cultivation,root rot,leaf ring rot and wilting have become a serious threat to the quality and yield of R.glutinosa.Biological control,as a green and environment-friendly control method,is widely used in the control of various plant diseases.In this study,a new strain of R.glutinosa leaf ring rot was isolated from R.glutinosa diseased leaf species,and its biological characteristics were investigated.We also screened a strain of Bacillus sp.y13 from endophytic bacteria of R.glutinosa,which has good inhibitory effect on both leaf ring rot and root rot pathogen,and studied its inhibitory effect and optimization of fermentation conditions to provide a theoretical basis for R.glutinosa disease control.The results of the study are as follows:（1）The pathogenic isolation of R.glutinosa leaves was carried out by tissue isolation method,and a total of 25 strains of bacteria were obtained.Based on the morphological characteristics of the colonies,the isolated strains were divided into three types,of which DHLW1 to DHLW15 were type I;DHLW16 to DHLW19 were type II;and DHLW20 to DHLW25 were type III,The strains DHLW2,DHLW16 and DHLW24 were selected from the three types of strains for the verification of Koch’s rule,DHLW2 was identified as the causative agent of R.glutinosa leaf ring rot.It is the first report of this pathogen causing ring rot on R.glutinosa.（2）The biological characteristics of Heterophoma verbasci-densiflori were analyzed and the results showed that:the optimum medium for this strain was potato dextrose agar medium（PDA）;it growed better under full light;it was not sensitive to p H;it couldan use maltose,fructose,lactose,soluble starch and peptone,glycine,potassium nitrate and ammonium sulfate as carbon and nitrogen sources;the best carbon sources was soluble starch and the best nitrogen sources was peptone;optimum growth temperature was 25°C;pathogenic mycelium lethality temperature is 52°C and pathogenic spores lethality temperature was 45°C.（3）Endophytic bacteria were isolated from healthy R.glutinosa leaves and tubers,and 18strains were screened for H.verbasci-densiflori DHLW2,H.novae-verbascicola DH1,Fusarium spp.DH2,and Rhizoctonia solani DH10,all of which have inhibitory effects on the four pathogens.Among them,y13 showed the best inhibitory effect on the four pathogens with74.43%,72.63%,60.38%and 58.63%,respectively.It was identified as Bacillus sp.by morphological and molecular identification.（4）The fermentation conditions optimization experiment was conducted for the screened Bacillus sp.y13 with high bacterial inhibitory effect.The optimal fermentation conditions were:soluble starch,beef paste+peptone and Fe SO₄.The optimized fermentation conditions were:1.38%soluble starch,2.83%beef paste and peptone,0.2%Fe SO₄,34.81°C,initial p H7,160 rpm,3%inoculum,and 100 m L loading volume per flask of 250 m L.The highest concentration of bacteriophage under this fermentation condition was 3.65×10⁸cfu/m L,which was 42.58%higher than that before optimization.