Breeding Of High-activity Serine Protease Strains And Their Biocontrol Effects

The reduction of agricultural area and rapid development of protective cultivation makes succession cropping commonly seen in agriculture.Soil-borne fungi and root-knot nematode diseases always occur in company,which enhances the infection and hazard.For high-efficient and sustainable agricultural development,it has become an urgent task to screen and breed bio-control strain,and produce anti-biological agent with high cell count.Serine protease is a protease that serine is contained in the active center.The existing research indicates that serine protease producing fungi are able to destroy body wall and ovi testa of root-knot nematode,and plays a positive role in controlling root-knot nematode.Baillus has been reported capable of controlling plant pathogenic fungi,but reports about bio-control effects on root-knot nematode of serine protease producing bacillus are rare.This research mainly aimed at screening serine protease producing bacillus to observe its bio-control effects on plant pathogenic fungi and root-knot nematode,selecting high enzyme activity strain as initial strain to discuss its cultivating and enzyme producing conditions,and obtain a mutant strain by through mutation and protoplast fusion.The main contents and results are as follow:1.Several serine protease producing strains have been isolated from materials like fermented black bean,fermented bean curd and tomato rhizosphere soil.BApNA,a special substrate for serine protease,and PMSF,specific inhibitor,were used to confirm the serine protease producing strains.SNUB 18 and SNUB 19 are two serine protease producing strains whose inhibition rates to PMSF were 77.86%and 91.20 respectively.By comparing the morphological characteristics,physiological-biochemical characteristics and 16S rDNA of the strains,SNUB 18 was classified as Xanthomonas campestris,and SNUB 19 was classified as Bacillus subtilis.Since anti-biological agent requires non-pathogenicity and long storage life,SNUB 19 was selected for further study.2.Antagonistic method,inhibition zone method and shake cultivation were introduced to observe anti-fungi effect of SNUB 19.It was discovered that the supernate of SNUB 19 was efficient in inhibiting turcicum PF3,Botrytis cinerea PF4,Grape anthracnose PF5,Pepper anthracnase PF6 and other plant pathogenic fungi.Meanwhile,it's capable of inhibiting spore germination and mycelial growth of Fusarum oxysporum.3.96-well plate was used to observe resist effect to nematode,the result showed that the sterilized filter liquid of SNUB 19 was relatively efficient in killing second instar larvae of root knot nematodes.4.Through L9(34)orthogonal array experiment design,the enzyme producing medium was optimized,the optimum ingredients are as follow,corn flour 40g/L,bean cake powder 10g/L,bran 10g/L,Na2HPO4 4g/L,KH2PO4 0.30g/L,Na2CO3 1.50g/L.After optimization,the enzyme activity was 132.1U,which was 58.63%higher compared to the activity before optimization.5.The optimum temperature of serine protease secreted by Bacillus subtilis SNUB 19 was 40 ?,when exposed in environment higher than 50? the enzyme activity decreased quickly.The optimum pH of the enzyme was 8.0 and was relatively stable between 7 and 8,when exposed in environment higher than 8.5,the enzyme activity decreased quickly.6.When discussing mutagenic effects of UV and NTG on SNUB 19,we found that UV could help get a high positive variation rate and large variation range.By using UV,mutant strain U-3-1 was screened,its serine protease activity was 184.89U,which was 29.06%higher than the initial strain.U-3-1 was then mutated by NTG,and obtained a mutant strain N-3-1,the serine protease activity of which was 215.78U and was 50.35%higher than 50.35%.7.Use UV to mutate SNUB 19,and screen by using agar medium which contained streptomycin and penicillin higher than lethal concentration.After secondary screen,mutant strains with high enzyme activity and drug resistance were obtained.Serine protease activity of P-1-5,a penicillin resistant strain,was 174.84U and was 1.29 times higher than its initial strain SNUB 19.Serine protease activity of S-2-2,a streptomycin resistant strain,was 167.26U and was 23.66%higher than its initial strain SNUB 19.8.Protoplast fusion was conducted with the material N-3-1,P-1-5 and S-2-2.After the fusion,H-3-11,a fusant with high serine protease activity and drug resistant marks was obtained.The enzyme was 273.67U,which was 1.65 times higher than the initial strain.The generation passage test showed that it was genetically stably.The innovative points of the passage are:(1)Serine protease producing Bacillus subtilis SNUB 19 was obtained and proved to be capable of controlling plant pathogenic fungi and root-knot nematode.Killing effects of serine protease producing Bacillus subtilis to larvae and ovum was found for the first time.(2)Through mutation and protoplast fusion,mutant strains and fusant were obtained.On account of the limited time,only one round of protoplast fusion was conducted,if the progressive multiple-parent protoplast fusion had been conducted,reshuffled strain with higher enzyme activity might have been gained.In addition,bio-control effects to plant pathogenic fungi and root-knot nematode for field crops still needs observation.