Screening Of Biocontrol Bacteria And Research On Control Effect Of Tobacco Brown Spot Disease

Tobacco brown spot,caused by Alternaria alternata,is one of the main diseases of tobacco.Since its discovery,it has seriously affected the yield and quality of tobacco,and brought huge economic losses to tobacco leaf production in China.At present,the most common control methods are using resistant cultivars and chemical pesticide.However,due to the limitations of cultivar and side effect of chemical agents,biological control has become the most promising management method for tobacco brown spot.Therefore,in this thesis,the antagonistic bacteria strains which have control effect on tobacco brown spot were screened through plate confrontation method,in vitro test,pot experiment and field experiment.And the optimization of culture conditions was carried out,so as to provide a theoretical basis for the development of biological control agents in the future.The results are as follows:1.In this thesis,58 strains of bacteria were isolated from the rhizosphere soil of healthy tobacco plants collected from fields with and without serious brown spot disease in the previous year.After screening by plate confrontation method,three antagonistic bacteria strains with inhibition rate of more than 50%against A.alternata were obtained,named T11,Y6 and Y2,respectively.Through the plate confrontation,it was found that the three strains also showed certain inhibitory effects on Rhizoctonia solani,Ralstonia solanacearum,Phytophthora nicotianae and Colletotrichum micotianae.The results showed that the inhibition rates on A.alternata of T11,Y6 and Y2 strains were 53.0%,59.6%and 50.0%respectively,when the ratio of antagonistic sterile fermentation filtrate and culture medium was 15%.The mycelial growth of A.alternata was still affected by the fermentation filtrate when the ratio was 5%.The mycelial expansion was found by microscopic examination as moniliform,apical enlargement,and nodular malformation hyphae,indicating growth inhibition.The physiological,biochemical and molecular identification of three antagonistic strains showed that the physiological and biochemical characteristics of T11 strain were close to that of Bacillus subtilis.Firstly,the 16S r DNA gene of T11 strain was detected,and T11 strain was identified as Bacillus.Then the gyr A gene of T11 strain was also detected,and the similarity of T11 gene sequence with that of B.subtilis strain srcm102751(CP028217.1)was 99%.Therefore,the strain was identified as B.subtilis.The physiological and biochemical identification of Y6 and Y2 strains was close to that of B.velezensis,and they were also classified as Bacillus by 16S r DNA gene detection.The results of gyr A gene detection showed that both strains were in the same branch with B.velezensis strain LF01(CP058216),and the reliability of this branch was 98%.Therefore,Y6 and Y2strains were identified as B.velezensis.2.The main culture conditions affecting the growth of strain T11 were determined by single factor and orthogonal test.Box Behnken regression test was carried out to optimize the three main culture conditions.The results showed that the best combination of culture conditions for strain T11 was:inoculation amount 2%,rotational speed 193 r/min,p H 7,liquid volume 110 m L,32.5?and culture for 22 h.The optimum medium composition was:beef extract 5 g/L,sucrose 15 g/L,and Mg SO₄·7H₂O 15 g/L.The optimal combination of culture conditions for Y6 strain was 3%inoculum volume,20 h culture time,30?culture temperature,108 m L liquid volume,193 r/min rotation speed and p H 6.6.The optimum medium composition was yeast extract 15 g/L,glucose 10 g/L,Mg SO₄·7H₂O 10 g/L.The optimal culture conditions for Y2 were as follows:inoculation amount 3%,culture time 18 h,temperature 30?,liquid volume 109 m L,rotational speed 202 r/min,and p H 6.8.The optimum medium composition was yeast extract 5 g/L,glucose 15 g/L,and Mg SO₄·7H₂O 5 g/L.3.The control effect of fermentation broth of three antagonistic strains showed that the average control effect of fermentation broth of T11,Y6 and Y2 were 55.78%,62.85%and 57.17%,respectively.In pot experiment,the average control efficiency of T11,Y6 and Y2 were 51.20%,68.18%and 59.81%,respectively.In the field plot experiment,in addition to 40%Junhejing,the best control effect was 10-time dilution of Y6 fermentation broth,whose relative control effect was 53.28%,followed by10-times dilution of T11 fermentation broth,with relative control effect at 44.5%.The10-time fermentation broth of Y2 strain also had a high effect,and the relative control effect was 40.76%.