| Fusarium wilt is a serious type of soil-borne disease.The main pathogen is Fusarium oxysporum,and blight can occur in a variety of plants.Fusarium wilt can occur during the entire growth period of the plant,and the damage is serious.There is currently no effective method for controlling blight.In order to reduce the microbial diversity in the soil,optimize the microbial community structure.And increase the microbial diversity in the soil,the plant can be inhibited by the wilt disease.Thereby reducing the occurrence of blight,and developing bio-inducing molecules and biocontrol bacteria as raw Anti-formulation,in this study,under the conditions of inoculation of Fusarium wilt pathogens,the mechanism of the use of biocontrol agents and the mechanism of controlling Fusarium wilt were studied.The main experimental research results are as follows:(1)In this paper,chitosan and seaweed polysaccharides were firstly selected,and their oligosaccharides were prepared by means of hydrogen peroxide and sodium hydroxide.The specific conditions were as follows:chitosan was treated with hydrochloric acid and hydrogen peroxide at 60°C for 6 hours to adjust the pH,three times the volume of ethanol precipitation.After drying,chitosan oligosaccharides were obtained;seaweed polysaccharide was treated with hydrogen peroxide at 90°C for 5hours,pH was adjusted,and algae oligosaccharides were prepared by drying at 50°C;Bacillus licheniformis A2-10 was optimized by shake flask optimization and single factor experiment.After optimal fermentation medium,the fermentation parameters were optimized by 20 L fermenter.Finally,the polyglutamic acid was obtained by a 500 L fermentor pilot test.The final fermentation conditions of polyglutamic acid are peptone2%,anhydrous calcium chloride 0.2%,maltodextrin 1%,anhydrous magnesium sulfate0.1%,sodium chloride 0.5%,sodium glutamate 5.4%,pH by ammonia The adjustment control is between 6.5 and 7.5,and the addition method supplements 12%of the total volume of 60%glucose solution,the polyglutamic acid content is 36 g/L,and the viscosity reaches 4900 mPa.s.Polyglutamic acid is optimized by heating to obtain oligoglutamic acid.Finally,the molecular weights of the three oligosaccharides extracted were determined by GPC,and the average molecular weight was 2310/1570/3650 Da,which belongs to the category of oligosaccharides.(2)In this paper,the strains of Trichoderma harzianum T22 B1-2 and Paecilomyces lilacinus B10-1 were selected and the spore powder was prepared by solid fermentation.Firstly,single factor and orthogonal optimization were carried out on fermentation temperature,material thickness,moisture and inoculum.The conditions were as follows:Trichoderma harzianum T22 B1-2 with cassava residue as the substrate,5%glucose as the carbon source,7%bean cake powder as the nitrogen source,the initial pH was controlled to 6,and the water content of the material was adjusted to 50%.The thickness of the control was 5 cm,and the inoculation amount of 8%was selected.The fermentation was carried out at 30°C for 7 days by means of shallow tray fermentation.The average sporulation of Trichoderma harzianum T22 B1-2 was 51.2×10~8 cfu/g.Paecilomyces lilacinus B10-1 with cassava slag as matrix,7%molasses as carbon source,5%bean cake powder as nitrogen source,control initial pH of 6,adjust material moisture content at50%,control The thickness of the charge was 7 cm,and the inoculation amount of 6%was selected.The fermentation was carried out by means of shallow tray fermentation at30°C for 7 days.The average sporulation of Paecilomyces lilacinus B10-1 was 27.5×10~8?/g.The final fermentation material was added with 5‰of trehalose and placed in a constant temperature drying oven for drying at a low temperature(30-40°C).The spore survival rate of Trichoderma harzianum T22 B1-2 and Paecilomyces lilacinus B10-1 was the highest.The blanks were increased by 37.29%and 37.81%,respectively,and mixed after pulverization to obtain spore powder.(3)Subsequently,we constructed a control evaluation scheme based on leaf infection.Firstly,a diseased fungus C1-1 was isolated and purified from the diseased banana plants.After molecular biological identification,a phylogenetic tree was constructed by using the neighboring method to prove C1-1 is Fusarium oxysporum,which is consistent with the reported main pathogens causing the disease of wilt.Next,it infects six leaves of American big leaf spinach,Tianqi,Wuzai,rapeseed,Chinese cabbage and oil wheat.Comparing the effects,the large-leaf spinach was selected according to the degree of infection;according to the infection effect,the culture time,temperature,infestation time and temperature of the pathogen were optimized,and the pathogen culture was finally determined for 5 days,the temperature was 30°C,and the infection time was 4 days.When the temperature is 28°C,the infection effect is the best.Next,the control effect of the bio-inducing molecule is screened based on the leaf infection experiment.The chitosan and dextran are finally screened according to the size of the lesion after infecting the leaf.The bio-inhibitory molecular inhibition rate was 38.26%and 37.32%(the inhibition rate after carbendazim treatment was 32.85%).(4)Furthermore,we used the model organism Arabidopsis to carry out the inducing effect and mechanism analysis of the attractant.Firstly,the infection model was established.The specific conditions were culture temperature 22°C,light 16 h,dark 8 h.Infection was carried out by adding 10 mL of fungus with a bacterium activity of 10~6-10~7cfu/mL to Fusarium oxysporum C1-1.The biocontrol bacteria were added to the roots by adding 10 mL of fungus to the roots 4-5 days before infection.10~7 cfu/mL of fungal liquid,bio-inducing molecules sprayed 10 mL by blade spraying.(5)Based on the constructed infection model,we carried out the transcriptional expression analysis of the key genes of the jasmonic acid pathway and the sodium salicylate pathway PDF1.2 and PR1,and extracted the Arabidopsis genome of the experimental conditions.After RT-PCR analysis,we found the dextran.The salicylic acid pathway PR1 gene was up-regulated 105-fold,and chitosan up-regulated the PDF1.2 gene of the jasmonic pathway 255-fold.This also confirms that the bio-inducing molecules based on chitosan and dextran are capable of activating the plant immune system based on leaf infection control.(6)Finally,we analyzed the effects of biocontrol strains on the structure of rhizosphere microflora in Arabidopsis thaliana.The results showed that the application of Trichoderma harzianum T22 B1-2 soil samples can significantly reduce the diversity and richness of diseased fungus.That is,the decrease in the content,including the decrease in the number of Fusarium oxysporum,and the obvious change in the structure of the flora.The application of soil samples of Paecilomyces lilacinus B10-1 and Trichoderma viride B1-1 reduced the diversity and abundance of fungal flora structure and increased the abundance and diversity of fungi,but most of them were pathogenic bacteria.And it also reduced the content of Fusarium oxysporum,which is not reflected in Arabidopsis.This result is consistent with the results of the biocontrol experiment.In summary,this paper explored the use of biocontrol agent and bio-inducing molecules to inhibit the growth of Fusarium oxysporum and control the occurrence of Fusarium wilt.The results of this study not only provide reference for biological control of blight,but also supplement the control methods of blight,which will help the development of the agricultural industry in the future. |
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