Isolation Of Antagonistic Bacteria Against Powdery Mildew Of Rosa Roxburghii Tratt And Biological Control Research

Rosa roxburghii Tratt has a unique taste of sour glutinous rice,high vitamin C content,mainly grown in the wild,and has certain cultivation and development value.At present,Guizhou,Sichuan and other regions have begun to establish large-scale establishment of Rosa roxburghii Tratt plantations.In the growth process of Rosa roxburghii Tratt,powdery mildew is one of the main diseases,which has a great influence on the growth,flowering and fruiting of Rosa roxburghii Tratt.At present,chemical germicides are mainly used for prevention and control,but such control measures can pollute the environment,endanger health,and cause disease-causing resistance.In contrast,biological control is a healthy and environmentally friendly new disease prevention and control measure.Therefore,it is hoped that antagonistic bacteria against thorny powdery mildew can be found,and the“green”color development of Rosa roxburghii Tratt industry can be realized from the source of cultivation.In this paper,the antagonistic bacteria against the powdery mildew of Rosa roxburghii Tratt were screened from its root soil and leaves,the antagonistic bacteria were identified from morphological,physiological,biochemical and molecular biology.The effect of spore germination indicates that it has good biocontrol potential;the culture solution is optimized for the biological control experiment of Rosa roxburghii Tratt,and the optimal concentration,dosage and spraying interval of antagonistic antibacterial control against Rosa roxburghii Tratt powdery mildew are clarified;The antagonism mechanism was explored in three aspects:the bacteriostatic enzyme,the bacteriostatic peptide,and the induction of the defense ability of Rosa roxburghii Tratt.The main contents and results are as follows:(1)Screening and identification of antagonistic bacteria against Rosa roxburghii Tratt powdery mildewThe microbial separation was carried out from healthy Rosa roxburghii Tratt and its root soil.The pathogen of Sphaerotheca pannosa was used as the target strain.The antagonistic bacteria were screened by leaf disc method.The bacteria were identified by morphology,physiological and biochemical characteristics and 16s rRNA.Their growth curves were plotted by measuring their OD600nm values.The results showed that JK-6 and JK-32 were significantly inhibited,and the control effects of leaf disc test were 60%and 57%,respectively.They were identified as Bacillus amyloliquefaciens and Bacillus aerius.Under the action of antagonistic bacteria,the spores of the pathogen of powdery mildew caused abnormal swelling and germination during the germination process.The surface of the mycelium became rough,swollen,entangled and melted,causing the cells to die,and the hyphae could not be differentiated to produce spores.The results of in vitro leaf control showed that JK-6had a good control effect on Rosa roxburghii Tratt powdery mildew,so the antagonistic bacteria JK-6 was further explored.(2)Optimization of growth conditions of antagonistic bacteria JK-6 and its antibacterial stabilitySingle factor optimization of carbon source,nitrogen source,NaCl concentration,temperature,pH,time,liquid volume and inoculum size 8 factors,PB test obtained temperature,pH,NaCl concentration three factors have a significant impact on JK-6growth,the steepest The gradient was determined by climbing,and the three-factor three-level response surface method was used to optimize the growth conditions of antagonistic bacteria JK-6.The optimal culture conditions were as follows:glucose 2g/mL,peptone 1 g/mL,NaCl concentration 0.8%,temperature 33.6?,pH 7.6,culture 24 h,liquid volume 40 mL,shaker speed 160 r?min-1.Under this condition,the OD600nm of the culture solution of the antagonistic antibacterial JK-6 was 1.418±0.002.The optimized culture solution of JK-6 was used to inhibit the germination stability of powdery mildew spores.The results showed that the culture solution had a stable inhibition effect on the germination rate of powdery mildew at pH=7 and temperature below 40?.And the tolerance of protease is higher;the culture solution maintains a high inhibitory effect on the germination of powdery mildew spores within one month after storage at room temperature 25±5?.(3)Biological control of antagonistic bacteria JK-6 on powdery mildew of Rosa roxburghii TrattIn order to clarify the control effect of JK-6 culture solution on Rosa roxburghii Tratt powdery mildew,the effects of different spraying concentration,dosage and interval time on the biological control of potted Rosa roxburghii Tratt seedlings were tested.The results showed that when the concentration of JK-6 culture supernatant was 109 CFU/mL,diluted 10 times,30 mL/pot,and the interval was 24 h,the control effect could reach 97.78%±0.43.(4)Preliminary study on the antibacterial mechanism of antagonistic bacteria against powdery mildew of Rosa roxburghii TrattAntagonistic antibacterial JK-6 production of bacteriostatic enzymes and antibacterial lipopeptides:antagonistic bacteria JK-6 can be metabolized to produce proteases,pectinase and cellulase.The menstrual blood plate and the oil drain ring test showed that JK-6 could be metabolized to produce surface active substances,and the genes of seven kinds of antibacterial lipopeptides in JK-6 were confirmed by PCR amplification test,namely itu A,itu A-1,Itu B,itu C,itu D,bamD and fen B-1indicate that JK-6 has the ability to metabolize the lipopeptides Iturin,Bacillomycin and Fengycin.Induction of antagonistic ability of antagonistic antibacterial JK-6 on the self-system of Rosa roxburghii Tratt:treatment with pathogenic bacteria(Spore suspension 10~5 cells/mL),antagonistic bacteria+pathogenic bacteria(1:1),and antagonism by spraying alone The bacteria(bacteria concentration 10~8 CUF/mL),the spray volume was 30 mL/pot,and the changes of the contents of various substances in the potted plants of the Rosa roxburghii Tratt pots after 6 different treatments were detected every 24 h.The test results showed that the comparison was only Spraying sterile water),the other three treatments will be peroxidase(POD),catalase(CAT),superoxide dismutase(SOD),phenylalanine ammonia lyase(PAL),and The activity and content of free proline(Pro)were induced.The antagonistic bacteria+pathogenic bacteria had the highest induction ability and the longest duration,and the peak value reached 23.57 U/g·min and 72.48 U/g,respectively.Min,214.04 U/g·min,25.36U/g·min;malondialdehyde(MDA)content recovered to a healthy level after 50 hours of treatment,and did not cause damage to plants due to the accumulation of MDA.In summary,the antagonistic bacteria JK-6 is metabolized to produce bacteriostatic enzymes and antibacterial lipopeptides,and induces the content of POD,CAT,SOD,PAL,Pro and other substances in the thorn pear to enhance its own defense ability and make the Rosa roxburghii Tratt powdery mildew is effectively inhibited.