RDNA-ITS Sequence Analyse Of Fusarium Oxysporum F.sp. Cubense Race1/race4 And The Establishment Of Transformation System Of Focr4

Fusarium wilt of banana caused by Fusarium oxysporum f.sp. cubense race1/race4, (Focr1/Focr4),is considered to be one of the most destructive diseases in banana production. Focr4 is most harmful to banana because it can infect all banana cultivars worldwide. The introduction and spread of Focr4 in China has become a major threat to the banana production in the country. To date there is neither an efficient chemical control method nor cultivars resistant to the disease. Since Fusarium wilt of banana is a soil-borne vascular disease, ordinary pesticides do not work efficiently, and the uncertain mechanism of pathogenicity has presented great difficulty to the breeding of resistant cultivars. rDNA-ITS(Internal transcribed spacer)regions of Focr1 and Focr4 isolates from Guangdong and Hainan provinces were analyzed in this paper. Common divergence was detected in their sequences, and may serve as a potential measure for rapid detection of physiologic races of Fusarium wilt. In addition, we established a transformation system of Focr4 based on Agrobacterium tumefaciens-mediated transformation (ATMT). An easy, rapid and efficient pathogenicity testing method in banana leaves in vitro was developed to select for non-pathogenic mutants. A molecular analysis of these mutants may lead to the cloning of the functional gene involved in pathogenicity and the clarification of the pathogenic mechanism. The results are briefly listed as follows:1. The result of pathogenicity test using Musa AAA and Musa ABB as hosts and PCR amplification using PBL/PBR,PCL/PDR as specific primers indicated that participant isolate Foc37(from Musa AAA) belonged to Focr 4, and Foc2(from Musa ABB) to Focr 1.2. rDNA-ITS regions of 3 Focr1 isolates and 5 Focr4 isolates were sequenced. The result showed that rDNA-ITS sequences of Focr 4 isolates and Focr 1 isolates had the same divergence in two positions: 366th and 385th bp. Focr 4 isolates all were T, but Focr 1 isolates all isolates were C.3. An easy, rapid, efficient and large-scale pathogenicity testing method in banana leaves in vitro was developed based on the pahtogenicity test on Focr1 and Focr4.4. On PDA culture medium, the mycelial growth of Foc4 was well inhibited at 150μg/ml of hygromycin. 5. The optimum conditions and parameters used in the establishment of transformation system of Focr4 were: A. tumefaciens incubated 6h in broth IM with 150μmol/L of AS before co-cultivation; A. tumefaciens (OD₆₀₀=0.15) broth culture and Focr4 spore(1×10⁶cfu/ml) suspension were mixed in equal volume and then co-cultivated for 48h.6. The results of the transformation experiment based on the above system are summarized here. For 1×10⁶ Focr4 conidia, an average of 150～200 of hygromycin B resistant colonies were obtained. With a pair of primers (Hf/Hr) designed by hygromycin phosphotransferase gene in plasmid pBHt2, a fragment of 819bp was amplified by PCR from all clones randomly selected from hygromycin B resistant colonies, the low false positive rate was confirmed. The transformants retained the hygromycin B resistance even after 6 consecutive transfers on medium without hygromycin B, thus proving the stability of hygromycin B resistance acquired.7. Through screening of the Focr4 T-DNA insertional mutants, we obtained a completely non-pathogenic mutant (Focr40321), 2 mutants with decreased pathogenicity (Focr40088 and Focr40003), and 14 mutants with different colony morphology and colony colour.