| As a characteristic aromatic plant and an important cash crop in China,Litsea cubeba has important uses in the spice and medical industries.Litsea cubeba leaf spot is a disease caused by fungi,which causes yellowing and wilting of infected plants and massive yield reduction and has a significant impact on the bilberry industry.Fusarium oxysporum is a global pathogenic fungus that can be transmitted through the soil and occupies an important position in plant pathology,being one of the ten most important pathogens.In this study,the pathogenic fungus was isolated and identified,followed by Dual RNA-seq analysis of the reciprocal transcriptomes of the host plants Litsea cubeba.and F.oxysporum,and functional validation of individual differentially expressed genes(DEGs)to enable us to better understand the mechanisms of their interactions.In-depth studies on the pathogenesis of F.oxysporum have laid the foundation for subsequent studies on the pathogenesis of the disease and drug sensitivity tests,which will help in the control of leaf spot disease of Litsea cubeba.The main research results are as follows:(1)The pathogenic fungi were isolated and identified.According to the morphological characteristics of the isolated pathogenic fungi,combined with molecular biology techniques and phylogenetic analysis,the pathogenicity test was performed using the Koch ’s rule.The pathogenic fungi were confirmed to be Fusarium oxysporum and preserved by conventional strain preservation methods.(2)The dual RNA-seq data of host plant Litsea cubeba and F.oxysporum were analyzed.KEGG and GO were used to annotate and enrich the functions of differentially expressed genes.A total of 671 differential genes were obtained from Foxy VS Dual,of which 350 were up-regulated and 321 were down-regulated.Litsea VS Dual obtained a total of 2074 differential genes,of which 1128 were up-regulated and 946 were down-regulated.(3)The differentially expressed genes were analyzed.In host plants,genes involved in defense response,such as antifungal compound secretion,ROS reactive oxygen species synthesis and defense signal transduction pathway,were significantly up-regulated.Genes encoding cell wall degrading enzymes,secondary metabolites and toxins,hormones and reactive Fusarium species production and extracellular proteases were significantly up-regulated in the pathogen,indicating that these genes are likely to play an important role in the infection of F.oxysporum on Litsea cubeba.(4)Through the comparison of interactive transcriptome data,it was found that in F.oxysporum,the alkaline protease FoALP was involved in multiple metabolic processes such as nitrogen compound metabolism,macromolecular metabolism,primary metabolism and organic matter metabolism.The protein has hydrolase activity,catalytic activity and serine peptidase activity,and may be involved in the pathogenic process of pathogenic fungi.(5)PEG-mediated genetic transformation of F.oxysporum.The protoplasts of F.oxysporum were successfully extracted,and the results of drug sensitivity test showed that the inhibitory concentration of hygromycin on mycelia and protoplasts of F.oxysporum was 50 μg/mL.In the study of the effects of different factors on protoplast preparation,the strain with the age of 3 days and the enzymolysis time of 3.5 h produced the largest number of protoplasts.(6)Functional verification of alkaline protease FoALP gene.FoALP gene can regulate fungal pathogenicity,which plays an important role in conidial germination,mycelial growth and response to external osmotic stress.This study will lay a foundation for further elucidating the infection of F.oxysporum and the immune mechanism of host.The implementation of this project will help to elucidate the mechanism of FoALP in fungal pathogenesis and may provide new targets for the prevention and control of fungal diseases. |
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