Studies On The Identification, Genetic Diversity Of Fusarium Oxysporum F. Sp. Momordicae And Differentially Proteomic Analysis On The Pathogen-Host Interaction

Fusarium wilt caused by Fusarium oxysporum f. sp. momordicae Sun & Huang (FOM) is one of the most serious soil-born diseases of bitter gourd (Momordica charantia L.). This disease has been widespreaded in bitter gourd planting regions around the world, including China, Philippines, Japan and India, and severely restricted the development of bitter gourd production. In this dissertation, the methodology of identification and genetic diversity of FOM were investigated, and a molecular detection method for this formae speciales was created. The resistance of bitter gourd germplasm to Fusarium wilt was evaluated, and the differentially proteomic analysis on pathogen-host interaction were also carried out. The main results were summarized as following.1. All of 48 isolates collected from six provinces of China during 2009-2010 were identified as FOM by the test of phenotype, pathogenicity and rDNA-ITS sequence. The pathogenicities of FOM isolates to bitter gourd seedlings were significantly different. The virulence of isolates to bitter gourd seedlings was not associated with the pigment of colonies. These isolates infected the seedlings of bitter gourd and some bottle gourd caltivars (Lagenaria siceraria var. Clavata, var. Depressa, and var. cougourda), but not those of var. gourda and other cucurbit species. The clustering analysis of rDNA-ITS sequence showed that 48 testing FOM isolates and 13 F. oxysporum f. spp. isolates from GenBank database were grouped into one cluster, whereas other Fusarium isolates were grouped into different clusters. However, the rDNA-ITS sequence could not distinguish the FOM isolates from other F. oxysporum f. spp. at formae speciales level.2. The genomic DNA polymorphism of 24 F. oxysporum f. spp. isolates was analysed by RAPD-PCR fingerprintings with 320 arbitrary primers. A unique 519 bp fragment S58-519 from FOM isolates was amplified. A pair of specific SCAR primers F1/R1 was designed to convert this RAPD marker S58.519 into SCAR marker RS.256. The SCAR marker was stable and selected for identifying FOM isolates. The detection sensitivity with this specific marker was 1 pg·μL-1 of pathogen genomic DNA, and 100 conidia per gram in soil. The SCAR-PCR based method could detect the FOM in the tissues of infecting bitter gourd seedling at early stage of disease development.3. The genetic diversity of 48 F. oxysporum spp. isolates from cucurbit was investigated using random amplified polymorphic DNA (RAPD) with 18 random primers. The average number of bands amplified by each primer was 14.5, and the average percentage of polymorphic loci was 97.81%. A cluster phenogram for 48 F. oxysporum spp. isolates was constructed with RAPD data. In RAPD dendrogram, all the FOM isolates were gathered into one phylogenetic branch (group G1) with the genetic similarity coefficient ranged from 0.92 to 1.00, which indicated that a high genetic similarity existed in FOM isolates, and classification of phylogenetic group was related to geographic origin in some extents.The genetic diversity of 48 FOM isolates from different geographical regions of China was characterized by amplified fragment length polymorphism (AFLP) analysis with 7 pairs of EcoRl-MseI primers. The average number of bands amplified by each primer pairs was 51.86, and the average percentage of polymorphic loci was 99.14%. In AFLP dendrogram, the groups were associated with the disease index of isolates. Group AG1 contained highly virulent isolates and their genetic similarity coefficients from 0.91 to 0.99. The subgroups of AG1 constituted with highly virulent isolates were highly relevant to geographical origin of these isolates. The other lower virulent isolates were clustered into eleven groups, showed significant genetic differences. The software PopGene was used to quantify the genetic divergence among the six geographical populations of FOM isolates. The results showed that it was low comparability degree and far genetic distance among populations. The genetic differentiation was mainly among populations and few gene exchanges. The parameters of genetic diversity indicated a high degree of genetic diversity in the FOM isolates.4. One hundred and forty-three inbred lines of bitter gourd were inoculated with FOM isolate AS-1 by root-dipping method at the seedling stage. Most of the germplasms were scored as susceptible or highly susceptible, out of which,8 germplasms were evaluated as resistant, and these resistant germplasms were collected from different regent of China, with characteristics of fruit as short club in shape, light green in skin color, stripe or grain-stripe in fruit ribbing. The germplasms from Southeast Asia country and Japan were susceptible to Fusarium wilt.5. Two bitter gourd germplasms, MC1-1-1 (resistant) and MC1-1 (susceptible) were inoculated with FOM by root-dipping method at the seedling stage and their differential expressions of root proteins were analyzed by two-Dimensional Gel Electrophoresis technology. Comparing with the control group, twenty-five differential protein spots, which changed more than 150% in protein abundance after inoculation, were found. In the 2-DE map of MC1-1,12 spots were up-regulated and 3 spots down-regulated. In the 2-DE map of MC1-1,4 spots were up-regulated and 6 spots down-regulated, respectively. Twenty-two protein spots were assayed by Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), and the peptide mass fingerprints (PMFs) were compared with the Mascot database. The identified 17 proteins including 8 proteins of cytophylaxis or radicals scavenging,4 proteins of basic metabolism,2 proteins of cell signal transduction,2 proteins of protein processing, and 1 proteins of photosynthesis, respectively.