Identification And Pathogenic Mechanism Of The Pathogen Causing Tomato Leaf Spot

At present,tomato disease is the main cause of the serious decline of tomato production,and brings huge economic loss to the tomato planting industry.From 2013 to 2014,when our research team investigated tomato diseases,a severe tomato leaf spot disease was observed in commercial tomato greenhouse in Dalian,Liaoning Province,China.This pathogen has the characteristics of fast infection rate and high mortality,which seriously affects the yield and quality of tomato in this area.The isolation and identification of pathogen causing tomato leaf spot,and the study of the invasion pathway andpathogenic mechanism of the pathogen infection of tomato plants are the important prerequisites for ccontrolling the disease.Thus,the research on this thesis is focused on the above scientific issues.The main research results and conclusions are as follows:(1)Our study found Fusarium proliferatum was the pathogen that caused tomato leaf spot.After collecting infected leaves of tomato leaf spot,the pathogen was isolated monospore and re-isolated from the infected tissues of the inoculated tomato plants to satisfy Koch's postulates.Finally,the causative pathogen was identified as Fusarium proliferatum(Matsush.)Nirenberg on the basis of morphological characteristics,culture characteristics and molecular biological characteristics.The taxonomic position of F.proliferatum is a fungus,which belongs to Ascomycota,Pezizomycotina,Sordariomycetes,Hypocreomycetidae,Nectriaceae,Fusarium,Gibberella fujikuroi D,Fusarium proliferatum(matsush.)Nirenberg.The strain has been named F.proliferatum K140108.(2)Fusarium proliferatum K140108 infected the tomato leaf tissue through stomata.Scanning electron microscope was used to observe the different stages of F.proliferatum K140108-infected tomato leaves.The results showed that the epidermal cells of tomato leaves became irregular and wrinkled when the F.proliferatum K140108-infected tomato leaves after 12 hous post inoculation(hpi).By 48 hpi,the spores were observed to enter the stomata.By 72 hpi,most of the stomata were filled out with spores.By 96 hpi,the stomata and epidermal cells of the tomato leaves were covered with spores,and the hyphae showed obvious growth on the stomata.At the same time,the brown necrotic spots had obvious appeared on the tomato leaves.(3)The interaction transcriptome of the F.proliferatum K140108 and tomato leaves based on RNA-seq technology.The leaf spot of tomato that infected by the conidia of the F.proliferatum K140108 with 96 hpi was used for transcriptome sequencing analysis,and mycelial stage was used as a control group.A total of 61,544,598 clean reads were de novo assembled to provide a F.proliferatum K140108 reference transcriptome.After the analysis of gene function annotation,a total of 75,044 unigenes were obtained,with 19.46%of the unigenes being assigned to 276 Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways,with 22.30%having a homology with genes F.fujikuroi.A total of 18,075 differentially expressed genes(DEGs)were identified,720 of which were found to code for secreted protein.Of these,184 were identified as candidate effectors,while 79.89%had up regulated expression.(4)After functional study of the five candidate effectors with significant up-regulated expression at 96 hpi,while the five candidate effectors were DN263,DN7752,DN12525,DN20100 and DN18029,the results showed that the five candidate effectors were not related to the pathogenicty of F.proliferatum K140108.In order to better study the function of the candidate effector genes of F.proliferatum K140108 by using the reverse genetic approaches,in this study,the preparation method of protoplast of F.proliferatum K140108 was optimized,and then we knocted out candidate effector genes by the method of split-PCR.Compared with wild type,the growing mycelium of ?dn7752 and ?dn12525 mutants were attached to the surface of the medium,Adn7752 mutant produced obvious purple pigment,Adn263,?dn20100 and ?dn18029 mutant phenotypes were no significant changes.The results of tomato leaf infection test indicated that the five candidate effectors had no obvious effect on the pathogenicity of F.proliferatum K140108.(5)The MAP Kinase FpPMK1 gene has a significant regulatory effect on the growth of mycelia and pathogenicity of F.proliferatum K140108.The F.graminearum GPMK1 gene was used to search for its ortholog in the F.proliferatum K140108 reference transcriptome.One DEG,DN56288,was identified to be highly similar to GPMK1 gene,and has been named FpPMK1.FpPMK1 gene shares 99%identity with GPMK1 gene of F.graminearum.After knockout FpPMK1 gene,the aerial hyphae of AFppmkl mutant became shorten,the growth rate and the yield of conidia of ?Fppmk1 mutant were significantly reduced.Tomato leaf pathogenicity test showed that,compared with wild type,the pathogenicity of AFppmkl mutant was almost completely lose the pathogenic ability of tomato leaves.The above results indicated that FpPMK1 gene plays an important role in regulating the pathogenicity of F.proliferatum K140108.The reversion test of knockout gene showed that the complemented strain of FpPMK1 gene could be restored in colony morphology,growth rate,conidia production and pathogenicity of tomato leaves.