Studies On Detection Technique Of Physiological Race Of Fusarium Oxysporum F.Sp.Cubense

Banana vasicular wilt often called as panama disease caused by Fusarium oxysporum f. sp. cubense(Foc) is world wide occurrence destructive disease to banana production. Race 4 of Foc was one of the pests in list of the object of plant quarantine in China. Rapid and accurate detection of Foc has important significance to control the spread of banana vasicular wilt. Development of detection technology apply facilitately for banana production,has the widespread significance in banana industry's production; Establish a rapid and accurate detection system have the positive sense regarding Chinese banana industry security.From 2006 to 2008, the main results obtained through the research were below:Fungal flora analysis found that F. solani, F. oxysporum var. redolens, F. semitectum, F. oxysporum, F. moniliforme and F. equiseti were the most common strain of Fusarium in the banana plants seedlings roots and field soil. Alignment of ITS sequence of Fusarium showed that determination of Fusarium rDNA-ITS sequences for rapid identification of Fusarium species was feasible. In addition, Cunninghamella,Paecilomyces, Trichoderma,Aspergillus and Fusarium were dominant fungi in banana plants seedlings roots and field soil.Developed a dual bacteriostasis KM differential medium for Foc 4 detection, results showed that the dual bacteriostasis KM differential medium proved to qualify for identifying Foc race 4 and low-cost and less demanding on the experimental conditions. FOCABB361 and FOCAAA315 were tested as standard strain for Foc physiologic race VCG detection to facilitate the application of detection in banana production.A new method for genomic DNA extraction of Foc conidiospore were development.The results indicated that the geonmic DNA extarcted from conidiospore with high purity and excellent repeatability. The geonmic DNA extarcted from conidiospore to conduct molecular biology research of Foc was more appropriate. Alignment of ITS sequence of Foc race1 and race 4 were performed on GenBank.The results indicateed that the full length of the ITS 1 of race1 and race 4 of Foc wer sharing 100 % identities and there were only 3 bp different in ITS2 sequence. Although differences of the pathogenicity were existent between Foc race 1 and race 4, there were no significant differences between the sequences of ribosomal DNA-ITS of Foc race 1 and race 4. The ITS sequence could not be used for the identification of races of Foc.RAPD analysis conditions of Foc were optimized to obtain optimal reaction system of RAPD-PCR ,and genetic diversity analysis of Foc showed that :Foc strains which isolated from the same region were at close relationships to one another.There were some differences of Foc race 1 and race 4 on the molecular level.Foc race 4 have more abundant genetic differentiation than Foc race1.Based on RAPD analysis of Foc strains , there was significant correlation between polymorphism and geographical origin of Foc strains.ITS-SCAR duplex PCR assay for detection of Foc race1 and race4 were developed.And the accuracy of detection results of ITS-SCAR duplex PCR assay were confirmed by the morphology and biology characters, differential hosts tests and rDNA-ITS sequence analysis. It was demonstrated that ITS-SCAR duplex PCR assay can be applied to non-significant disease banana plants seedlings and field detection. Using ITS-SCAR duplex PCR assay,at one PCR amplification Foc race1 and race4 can be detected simultaneously. It overcame the shortcoming of general PCR and demonstrated that ITS-SCAR duplex PCR assay can make a positive diagnosis when banana plants infected by Foc but did not show symptoms and saving a great deal of time.ITS-SCAR duplex PCR assay can be used as a powerful tool for the monitoring and detection of Foc.