The Establishment Of The Molecular Detection Technique And Basic Identification Of Effectors Of Fusarium Oxysporum F.sp.niveum

Currently, the disease of watermelon wilt is controlled significantly by chemical fungicides. But the chemical fungicides always intend to leave residues on plants, destroy the ecological environments and put health of people at desperate risks. Therefore, the vital precondition of inhibiting the disease of watermelon wilt effectively is to enhance the process of unravelling the mechanism of pathopoiesis of the disease. The effector genes play a pivotal role in the process of the pathogens invading the plants. Therefore, the primary research of effector genes could be helpful in elucidating the pathogenic mechanism of the pathogens on a molecular level so as to provide theoretical basis for the control of watermelon wilt.The research includes the following contents:1. The data of whole genomes of 3 physiological races were analysed on the basis of sequencing and resequencing of the whole genome of Fusarium oxysporum f.sp. niveum. And 115 fragments of specific genes of FOW were screened out through bioinformatics analysis. The primers were designed based on the 2 longest fragments of genes. FOW-2F / FOW-2R、FOW-3F / FOW-3R、FOW-4F / FOW-4R and FOW-6F/ FOW-6R primers were screened out to be utilised in conventional PCR system so as to detect rapidly and accurately FOW races. The specific primers of and common primers of conbined together, we established a duplex PCR method which could also achive the goal of detecting FOW races rapidly and accurately.2. The genomes were assembled and interpreted based on sequencing of the whole genome. Among which, N50 of FOW1 reached 2.16 M.137 secreted proteins were screened out through the analysis of bioinformatics. Comparative genomic analysis were carried out among FOW0、FOW1and FOW2 with BLAST,and the 27 effector proteins were identified. Among which, 7 effector proteins were influenced by positive selective-pressure,as per the caculation of Ka_Ks_Caculator software.3. The standard strain FOW1 A was utilised to infect the susceptibe varity of watermelon and the RNA of roots of invaded watermelon plants were extracted at pre-inoculation and post-inoculation stages, and transcriptome sequencing was carried out.The 14 genes of differential expression were identified based on the analysis of genomic and transcriptome and comparision of homologous genes from Cs-20, cucumber and melon, combined together.4. After q RT-PCR analysis, expressions of FOW1A₀2929、FOW1A₁0400、FOW1A₀8150、FOW1A₁2121 and FOW1A₀5310 effector genes were obviously up-regulated, which may be the pathogenic effectors.In conclusion, a duplex PCR method was used to rapidly detect FOW; Pathogenesis-related proteins of FOW1 was analysed, and candidated effectors were predicted through comparative genome analysis; Candidated effectors of FOW1 A were predicted through genome analysis and transcriptome analysis, which were tested through q RT-PCR. These studies will provide theoretical basis for the diagnosis of diseases in the watermelon growing fields and prevention of FOW.