Screening Of Anti-fungus And Optimization Of Fermentation Conditions For Tobacco Brown Spot Disease

Tobacco brown spot disease is one of the main diseases in tobacco,which causes huge economic loss to our country's tobacco production every year.At present,there are many methods for prevention and control of brown spot disease,and which,the most promising and potential method is biocontrol.Tobacco rhizosphere soils and leaf circumferences of tobacco leaves contain a large number of microbial resources,and some microorganisms are widely used in biological control because of their characteristics such as inhibiting pathogenic microorganisms,promoting tobacco growth and inducing resistance of the system.In this paper,82 strains of bacteria were isolated from tobacco rhizosphere soils,and the bacterial F11 strain with strong antagonism to tobacco brown spot pathogen was screened by the plate confrontation method and the double layer plate method,and the aseptic fermentation broth was used to the tobacco brown spot pathogen(Alternaria alternata)of the inhibitation band reached up 23.5 mm.The results showed that the physiological and biochemical characteristics of F11 strain were the closest to Bacillus methylotrophicus,and the results of of 16 S r RNA gene sequence and the genus Bacillus were 99% homology.The gyr A gene of F11 strain was detected,and the gyr A gene sequence of F11 was homologous to the gene sequence of Bacillus methylotrophicus Y37 gyr A strain,So the strain was identified as a B.methylotrophicus.The best medium was screened out from 5 kinds of common bacteria medium.Using the Design-expert software,the response surface method was used to find the optimal fermentation conditions.The best culture medium is PDB,the best conditions are: Culture temperature 35 ?,initial p H 7.5,inoculation amount 3%,fermentation time 36 h,shaking bed rotational speed 160 r/min,and bottled quantity 100 ml/250 ml.Under the optimum conditions,the inhibitation zone of fermentation broth can reach up 26.8 mm,which is 32% higher than that(20.3 mm)before optimization.This strain has strong antifungal activity,has the potential of developing the biological inhibitor,and provides some references for the study of the mechanism of tobacco brown spot disease.The preliminary study on the mechanism of the biological control of the bacteria showed that:(1)The pathogenic fungal hypha of caused the tobacco brown spot growing in the fermentation liquid of F11 was obviously affected,the main features showed the apical enlargement,the hypha deformity forming nodular,the protoplasm obviously gathering,the hypha bead likely inflating,the cells rupturing and the cytoplasm extravasation.(2)Fermentation supernatant of F11 under the different concentration can inhibit spore germination of tobacco brown spot pathogen,and the inhibition rate at different time increased with the increase of supernatant concentration in fermentation.The spores of tobacco brown spot disease treated by different concentrations was characterized by vacuole and rupture,cytoplasmic extravasation,abnormal germination,and swelling and bubbly in the germination area,which could not develop into normal hyphae.(3)After treatment of the F11 fermentation supernatant,the electrolyte leakage occurred seriously,and the conductivity of the treatment was higher than that of the control.(4)The contents(soluble sugar and soluble protein)of the tobacco chain mycelia treated by F11 fermentation were largely extravasation,and the content of total sugar and soluble protein was lower than that of the control group,which resulted in the decrease of the total sugar and soluble protein in the mycelium.(5)The activity of Malic Dehydrogenase(MDH)and Succinic Dehydrogenase(SDH)decreased after treatment the F11 fermentation supernatant,which resulted in slow or stagnant growth of mycelia.The field control test was carried out by using 10% difenoconazole,40% dimethachlon,10% polyoxin,F11 strain fermentation supernatant,and F11 strain active bacteria solution.The results showed that 3 kinds of medicament treatment and 2 kinds of fungicides had some preventive and therapeutic effects on tobacco brown spot disease.One of the best prevention effect is more than 10% polyoxin,the relative control effects is 63.14%,followed by 40% dimethachlon,the relative control effect of 58.71%;10% difenoconazole,the relative control effect is 49.71%,both the ferment supernatant of F11 strain and the F11 strain had better control effect,and the relative control effect was 41.43% and 49.86%,respectively.