Antagonism Against Plant Pathogenic Fungi And Biocontrol Of Tobacco Brown Spot By Bacillus Velezensis HY19

Pathogenic fungi infect plants and cause fungal diseases,which is one of the main reasons for crop yield reduction.Chemical control is the simplest and most economical and effective method to prevent and control numerous fungal diseases in modern agriculture.However,long-term use of chemical pesticides on a large scale has caused a series of safety,production and environmental problems.Due to safety and harmlessness,it is necessary to develop safe,powerful,and broadspectrum resistant bio-pesticides.In the present experiments,thesis author studied the basic biocharacteristics,antifungal activity,and control efficiency of selves-isolated Bacillus velezensis HY19 against plant fungal pathogens,flue-cured tobacco brown spot and mechanism of disease resistance.Following are the main results obtained.1.This selves-isolated bacterium is gram-positive and rod shaped((0.89-1.23)×(0.53-0.63)μm in length),endophytic spores.The colonies were white in color,and opaque in transparency with irregular edges and dry and wrinkled surfaces.They grew well in nutrient agar containing salt less than 7% at p H5-8 and 20-36℃,and was able to produce endophytic spores and a variety of hydrolytic enzymes(including protease,amylase,phosphatase,and lipase).Based on the morphology,physiological and biochemical characteristics,and 16 S r DNA sequence,this bacterium was identified as Bacillus velezensis designed as strain HY19(hereinafter referred to as HY19).2.HY19 exhibited antagonistic activities against a variety of plant pathogenic fungi to varying degrees,including Colletotrichum gloeosporioides,Mycosphaerlla melonis,Alteraria alternata,Sclerotinia sclerotiorum,Rhizoctonia solani,Bipolaris sorokiniana,Phytophthora capsici,Phytophtora parasitica,and Botrytis cinerea.The inhibitory rateranged form 56.75(Phytophtora parasitica)to 100%(Sclerotinia sclerotiorum).The mycelial deformity,including more beaches,swollenness,and tortuosity occurred when the pathogen met HY19 on the agar mediums.3.HY19 fermentation broth also showed antifungal activities against the tested pathogenic fungi.The inhibition efficacies increased with the concentrations,which were 56.41-81.60% for Alternaria alternata,20.84-50.55% for Mycosphaerlla melonis,and 22.82-46.43% for Colletotrichum gloeosporioides when grown on the agar medium containing 5,10 and 20% of the fermentation broth.The volatile substances produced by HY19 also inhibited the growth of the the three pathogenic fungi above mentioned,with the inhibitory rates of 33.13% for Alteraria alternata,27.66% for Fusarium oxysporum,and 32.19% for Colletotrichum gloeosporoides.Additionally,it is worth noting that the anti-activities of the fermentation broth against the pathogen fungi were acid-base and heat stable.For example,the antifungal activity changed little,if any at all,after heating for 30 minutes at 70-100℃and maintain at p H 3-11 for 24 hours.4.The Alternaria alternata treated with HY19 fermentation broth and volatile gas reduced the protein content,superoxide dismutase(SOD),catalase(CAT)activities and increased the malondialdehyde(MDA)content significantly in mycelium.The fermentation broth can significantly inhibit the germination of spores and the elongation of the germ tube.Transcriptome sequencing analysis showed that after treating Alternaria alternata with HY19 fermentation broth,2112 genes were differentially expressed in the pathogenic fungi(1157 genes were up-regulated,and 955 genes were down-regulated),it inhibited the metabolism of sucrose and starch,hindered the glucose entering into the glycolysis pathway,thus inhibited the utilization of carbon source and energy supply in the pathogen,resulting in poor growth and even death of the pathogen.5.In the potted control test of flue-cured tobacco brown spot disease,the prevention and treatment effects of HY19 fermentation broth were not significantly different from those of chemical pesticides(polyoxin).The prevention and treatment effects were 69.31% and 72.72%,respectively.Compared with the treatment only inoculated with pathogenic fungi,HY19 prevention and treatment can significantly reduced the malondialdehyde content and relative conductivity,increased activity of superoxide dismutase(SOD)and peroxidase(POD)in flue-cured tobacco leaves.