Control Effect Of Three Bacillus On Tobacco Black Shank And Its Biocontrol Characteristics

Tobacco black shank is a rhizome disease caused by Phytophthora parasitica var.Nicotianae.It is an important factor restricting the quality and safety of tobacco production.Control measures for tobacco black shank include disease-resistant varieties,chemical control and biological control.However,it is difficult to cultivate disease-resistant varieties and there are few applicable areas,long-term large-scale use of chemical pesticides is likely to cause pesticide residues,environmental pollution and drug resistance.With the proposal of green prevention and control concepts,environmentally friendly and efficient biocontrol bacteria have become the prevention of tobacco black the main force of shin disease.Therefore,i hopes to control tobacco black shank disease through biological control measures,and research the biocontrol characteristics of the strains.In order to solve the problem of unstable antibacterial activity of single biocontrol bacteria,we screened a composite strain with high antagonistic activity against Phytophthora parasitica var.nicotianae from six biocontrol bacteria?GY1?GY5?GY8?GY9?GY10?GY12?by compatible test,antagonist test and pot test.Clarify the taxonomic status of strains through molecular biology.The antibacterial spectrum,secondary metabolites,volatile substances,rhizosphere colonization ability and growth-promoting ability were studied by compatible test,a double petri dish assay and pot test.The main results are as follows:1.Through the antibacterial test,compatibility determination and pot test,it is found that GY1,GY10and GY12 have high antibacterial rate and good compatibility.The results showed that the mixed application of GY1,GY10 and GY12 after fermentation had the best control effect on tobacco black shank,and the plate inhibition rate reached 86.9%,the potted control effect reached 74.53%,which was increased by 15.34%,27.42%and 44.94%compared with single strains.2.The 16S rRNA gene and gyrA gene sequences of biocontrol bacteria were determined,and the phylogenetic tree was constructed by the adjacency method.Molecular identification showed that GY1,GY10 and GY12 were Bacillus velezensis,Bacillus amyloliquefaciens and Bacillus subtilis,respectively.3.Optimized the medium with single factor test and orthogonal test.The optimal fermentation mediums of GY1,GY10,and GY12 were obtained by screening.GY1:corn flour 10 g/L,yeast extract power 10 g/L,NaCl 5 g/L;GY10 and GY12:corn flour 10 g/L,peptone 10 g/L,CaCO₃ 5 g/L.After 24hours of fermentation,the biomass increased by 42.63%,51.90%and 9.05%compared with NB.The results showd that the combination of GY1,GY10 and GY12 can improve the control effect.4.Observe the germination rate after immersing seeds in the bacterial suspension to determine whether the bacterial suspension can promote seed germination.Root irrigation was used to detect its growth promoting effect on tobacco.The results showed that GY1,GY10,and GY12 could increase the germination rate of tobacco seeds.GY1,GY10,and GY12 had the best effect when the concentration of the bacterial suspension was 8×10⁷ cfu/mL,6×10⁷ cfu/mL,6×10⁷ cfu/mL.Root irrigation after compounding can promote the growth of tobacco.Compared with the control,the plant height,stem circumference,dry weight,wet weight and root-to-root ratio were significantly improved.The growth promotion rates were 14.01%,22.75%,77.19%,83.14%and 14.29%,respectively.This showd that choosing the right medium can improve the fermentation efficiency.5.Use biochemical analysis methods to further understand the mechanism of disease prevention and growth promotion of GY1,GY10 and GY12.It was found that the 3 strains not only have a growth promoting effect,but also have a broad spectrum of bacteriostasis,which can inhibit 8 fungal diseases such as Alternaria alternata.Sterile fermentation broth has a significant inhibitory effect on Alternaria alternata.Volatile substances have significant inhibitory effects on Phytophthora nicotianae and Alternaria alternata.3 strains can produce ferrophiles,proteases,amylase and other substances.GY1,GY10,and GY12 protease inhibitory substances can inhibit the growth of tobacco brown spot pathogen,and the antibacterial activity decreases under high temperature,acidic,and alkaline environments,but still has a certain tolerance to adverse environments.6.The colonization ability of strains GY1,GY10,and GY12 in the root soil of tobacco plants was determined by the natural antibiotic labeling method.The results showed that the resistance markers did not affect their growth and bacteriostatic ability,and all three strains could be determined in the tobacco rhizosphere soil.Compared with a single strain,GY1-10-12 colonization in the rhizosphere of tobacco increased.Therefore,it is speculated that the 3 strains may play a role in disease prevention and growth promotion through the above characteristics.