| Wheat common root rot is one of the main causes of wheat yield loss and quality degradation.Hebei province is an important area in China for the yield of wheat.The wheat production is mainly kepted by chemical prevention and controlling for the less of resistance varieties to Bipolaris sorokiniana.However,with the emergence of pathogen with resistance to pesticides and the gradual improvement of people’s understanding for environment and food safety,biological controlling has become a more promising method for disease controlling and meeting the requirements of modern people.In this study,a biocontrol strain with strong antagonism to wheat common root rot has been got based on screening the antagonist bacteria,which saved in my laboratory.The strain was identified to determine the status of taxonomic,was optimized the medium composition and culture conditions,was verified the colonization ability in the soil and plants,and was preliminarily identified the bacteriostatic substances,so as to provide a basis for the development of biological control agents in the future.The main results as follows:1.36 strains of antagonistic bacteria stored in the laboratory were screened for antagonistic strains to B.sorokiniana,Fusarium pseudograminearum,F.graminearum and Rhizoctonia cerealis by the plate confrontation culture method.The results show that the strain K-8 has a strong antagonistic effect on B.sorokiniana and also effects on the remaining three pathogens.2.The K-8 strain was determined to be Bacillus amyloliquefaciens based on the morphological,physiological and biochemical characteristics,and molecular characteristics.Single colony nearly round,regular edge,milky colours without pigment yielded,G+,positive denitrification,positive gelatin liquefaction test and negative MR test,etc.The homologous sequences of 16SrDNA and Gyrb with the standard strain DSM7(Bacillus amyloliquefaciens)are up to 100%.3.The optimization of culture medium composition of K-8 was improved on the basis of LB medium through single factor test and orthogonal optimization design test,the B.sorokiniana as the pathogenic target pathogen,the bacteria concentration and bacteriostatic effect as the indexes of bacteriostatic performance.The most suitable medium and culture conditions were soluble starch 1%,beef extract 0.5%and manganese sulfate 1%,cultured for 48 h at 35℃ with liquid loading of 50 mL/250 mL,inoculum quantity of 2.00%,rotation speed with 200 rpm.4.The mutant strain K-8SR with resistant to high concentration of streptomycin and rifampicin was screened by step mutagenesis.The strain K-8SR was mixed into the sterilized soil to detect the colonization in soil and plants.The number of bacterial colonies in the 2nd rhizosphere soil and root topsoil reached the maximum,1.80×105 CFU/mL and 1.50×105 CFU/mL respectively,and the lowest number 0.00 × 105 CFU/mL and 0.03 × 105 CFU/mL on the 42nd day,respectively.On the 2nd day,the labeled strain had invaded into the root,and the colony number was 0.54×105 CFU/mL.The maximum colony number was 0.84×105 CFU/mLon the 12th day.The minimum number of colonies on the 42nd day was only 0.07 × 105 CFU/mL.The K-8SR strain was isolated from stem on the 3rd day wiht colonies number 0.65 × 105 CFU/mL,and with the maximum number 0.97 × 105 CFU/mL on the 7th day.Tthe amount of bacteria is significantly reduced on the 36th day with the colonies number 0.01 × 105 CFU/mL.The K-8SR strain was isolated from leaves with colonies number 0.01 × 105 CFU/mL.The maximum number of colonies was 0.71 × 105 CFU/mL on the 12th day,and the colonies number was 0.02 × 105 CFU/mL on the 42nd day.During the whole test period,the colonization of the labeled strain with a trend of go up and then fall.5.The extracted small molecular metabolites in the aqueous phase parts and n-butanol,which was extracted with different polar organic solvents from the sterile fermentation broth of antagonistic strain K-8,had different degrees of bacteriostatic effect on wheat root rot fungus.The 75%saturation ammonium sulfate crude protein extraction has the best bacteriostatic effect with bacteriostatic rate 82.21%,indicating that the bacteriostatic protein may be the main active substance.The bacteriostatic effect of antibacterial protein is stable for a bacteriostatic rate of about 80.49%after treated at 4-100℃ for 30 min,pH 3-10 for 30 min and ultraviolet irradiation for 36 h.6.The antifungal effects of volatiles produced by K-8 strain against the pathogen of wheat common root rot were conduceted by inverted plate method.After 7 days of cultivation,no obvious antibacterial effect was found,the colonies of wheat common root rot were gray and black,and the aerial mycelium were luxuriant and felted.However,the volatiles were extraced with petroleum ether,and the bacteriostatic rate was 11.35%,which indicating it is weak bacteriostatic activity. |
