Studies On Screening And Preliminary Mechanism Of Biocontrol Bacillus On Pepper Anthracnose

Capsicum annuum L.is a solanaceous crop with important economic and medicinal value.Guizhou’s pepper output ranks first in China.With the increase of Guizhou’s pepper planting area and the growth of years,field diseases have become increasingly serious.At present,pepper production mainly relies on chemical pesticides to protect health,resulting in a series of negative effects,such as the "3R" problem of pesticides.Therefore,biological control of environmentally friendly plant diseases has attracted increasing attention.Therefore,this study took pepper anthracnose in Huaxi District of Guiyang City as the research object to determine the type of pathogenic bacteria,screened the beneficial bacillus to prevent and control the disease through the plate confrontation method,indoor pot planting,and field plot test,and initially studied the biocontrol mechanism to provide a theoretical basis for the development of Guizhou’s research and development of biological agents for controlling pepper anthracnose.The main findings are as follows:1.Pathogen identification.Through the isolation,purification,pathogenicity determination,morphological and molecular biological identification of culturable pathogens,the pathogen of capsicum anthracnose in Huaxi District of Guizhou Province was identified as Colletotrichum scovillei.2.Screening and Identification of Biocontrol Bacillus.Strains AL210,MT327,and MT332 have strong inhibitory effects on pepper anthracnose,and the inhibitory bandwidths are 21.0,19.2,and 17.8 mm,respectively.The three strains also had strong antifungal activity against 5 important plant pathogenic fungus such as Alternaria alternata,Phytophthora nicotianae,Phomopsis sp.,Sclerotinia sclerotiorum and Magnaporthe oryzae.By physiological and biochemical detection and 16 S rRNA and gyr A gene sequence comparison,strains AL210 and MT327 were identified as Bacillus velezensis,and MT332 was B.subtilis.3.Indoor bioassay and field plot prevention test.The results of in vitro fruit control experiments showed that the pepper fruits treated with different biological control bacillus AL210,MT327 and MT332 showed a certain degree of control effect on pepper anthracnose.The results of indoor pot experiments showed that the three strains have good control effect on pepper anthracnose,the best control effect of MT327 is 72.40%,the control effect of AL210 and MT332 is 43.18% and 65.91% respectively,MT327 also has obvious effect on pepper Promoting effect.The results of field tests showed that all three strains were effective against pepper anthracnose.and the control effect of MT327 is relatively better.The control effects on the 10 th and 20 th days after the treatment are 53.95% and 55.10%,respectively.4.Biological characteristics of biocontrol bacteria and determination of biocontrol related enzymes.In this study,the AL210,MT327 and MT3323 strains all entered a stable growth period in 7.0-8.0 h,and were able to inhibit a variety of plant pathogenic fungi,resulting in swelling and deformity of the pathogenic mycelia;all three strains had strong Exercise ability;can produce more complex biofilm structure,protease,cellulase,ferrophil,phosphatase and other biocontrol related enzymes.In genetic transformation experiments,AL210 and MT332 can successfully transform p GFP78 plasmid.Electron microscope scanning and laser confocal microscope observation showed that AL210 can effectively colonize the roots of pepper plants.5.The chemotactic effect of Bacillus on pepper root exudates.Collecting pepper root exudates using a vacuum freeze-drying instrument for GC-MS non-target omics detection,there are 43 substances with a similarity above 69%,of which organic acid substances account for the main proportion,a total of 27;amino acids 8 Species;alcohols,amines,esters are few.Six of these components(phthalic acid,lauric acid,nicotine,cinnamic acid,fumaric acid,and benzoic acid)were screened to determine the chemotaxis of AL210 to them.The results showed that AL210 showed varying degrees of chemotaxis to the six substances reaction.6.Determination of key enzyme activity for induced resistance.This study measured changes in PAL,POD,and PPO enzyme activities in pepper leaves treated with AL210,MT327,and MT332.The results showed that the activities of PAL,POD,and PPO in the leaves of peppers treated with different biocontrol bacteria significantly changed with time,and the enzyme activity performance was slightly different between different treatments at the same time point,indicating that pepper resistance to anthracnose and PAL after Bacillus treatment,POD,PPO activity are closely related.7.AL210 whole gene sequencing analysis.In this study,B.velezensis AL210 was obtained by sequencing the whole genome.It consists of a circular chromosome with a size of 4 011 788 bp,and the GC content is 47.2%.The AL210 genome contains 3965 protein coding sequences(CDS),accounting for approximately 89.8% of the genome.There were 87 predicted tRNAs and 27 rRNAs.