Construction Of The Fluorescent Protein Labeling System Of Trichoderma Viride And Its Biocontrol Effect On American Ginseng Root Rot

American ginseng root rot is a soil-borne disease,which has a serious impact on the cultivation of American ginseng.As an important biocontrol fungus,Trichoderma has an important contribution in the field of biological control.In the preliminary research in the laboratory,it was found that(Trichoderma atroviride)HB20111 has a good control effect on the disease.Therefore,this paper takes Trichoderma viride as the research object.On the one hand,it uses molecular labeling technology to transform the Trichoderma viride strain,making it a tool for us to "visualize".On the other hand,through high-throughput sequencing,from the perspective of soil microbial community,explore the impact of the application of Trichod erma viride on the rhizosphere soil microbial community of American ginseng,and explore the correlation of Trichoderma viride HB20111 against root rot from an ecological point Mechanism to provide a theoretical basis for biological control of American ginseng root rot.The results obtained by the experiment are as follows:(1)Take the binary expression plasmid of plant and Agrobacterium(p CAMBIA1301)as the mother vector,introduce the Hyg expression box,Trcp promoter and terminator derived from the fungus RNAi gene silencing interference plasmid(p Silent-1)at the point of polyclonal sequence.The filamentous fungal expression vector P-Trcp-HYG-Ds Red-GPDA was constructed,and then the red fluorescent protein gene-containing plasmid p CMV-C-Ds Red was used as a template to clone and obtain the red fluorescent protein gene sequence,which was inserted into the vector multiple cloning site as a report Gene;At the same time,the fungal gpd A promoter(derived from p AN8-1-gpd A)was introduced at the N-terminus of the red fluorescent protein.Through the Agrobacterium-mediated genetic transformation method,a dark green Trichoderma strain expressing the red fluorescent protein was obtained.The interaction study of Trichoderma HB20111 with plants or plant pathogens provides a powerful technical tool.(2)Agrobacterium-mediated transformation was used to realize the transformation and expression of the tool vector in Trichoderma virens,and the transformed strain of Trichoderma with red fluorescent protein expression was observed under a fluorescent microscope.In the transformation process,the experiment determined that 150?g / m L hygromycin B was used as the screening concentration for transformants,and 150?g / m L cephalosporin was used as the antibacterial concentration of Agrobacterium;orthogonal test optimized the transformation method of Agrobacterium,The results found that we determined that the best experimental conditions were Agrobacterium OD600 0.8,co-cultivation time 36 h and spore concentration 106 / m L,the conversion efficiency was the highest,we carried out the experiment according to the best conversion conditions;The transformation efficiency of Agrobacterium tumefaciens EHA105,LBA4404 and AGL1 against Trichoderma virens was found to be the best Agrobacterium tumefaciens type for the transformation of Trichoderma virens HB20111 with EHA105.The final transformation efficiency reached about 110 transformants / 106 Trichoderma spores.(3)In the field experiment,the effect of inoculation with Trichoderma viride on the germination rate of American ginseng and the incidence of American ginseng root rot was investigated;the experimental results found that the germination rate of American ginseng in the experimental group inoculated with Trichoderma was higher than that in the control group In controlling the incidence of American ginseng root rot,inoculation with Trichoderma virens can effectively prevent and control root rot,and its control effect is as high as 74.2%;through high-throughput sequencing and modern bioinformatics methods,inoculation with Trichoderma virens was analyzed The effect on the microbial community structure and abundance of American ginseng rhizosphere.The results showed that: Trichoderma inoculum inoculation had little ef fect on the fungal and bacterial microbial diversity of American ginseng rhizosphere;however,in the rhizosphere,American ginseng root inoculated with Trichoderma The structure and abundance of fungal and bacterial communities in the world changed signif icantly;through fungal function prediction,it was found that inoculation with Trichoderma viride significantly reduced the relative abundance of plant pathogens in the rhizosphere community of American ginseng,through the interaction between microorganisms The network diagram found that Trichoderma had a negative correlation with various plant pathogens including Fusarium oxysporum root rot pathogen.