The Viability And AFLP Analysis Of Verticillium Dahliae Preserved In Mineral Oil At Room Temperature

Verticillium wilt of cotton, caused by Verticillium dahliae, is one of soilborne diseases. The disease distributed all over the world is one of the most important factors limit the cotton productuction. In our lab, almost 2000 strains of plant pathogenic fungi, including V. dahliae, V. nigrescens, and Fusarium oxysporum f.sp. vasinfectum, were preserved in mineral oil at room temperature. In this thesis, 261 strains of plant pathogenic fungi preserved in mineral oil were recovered to test the viability through PDA plate method. The basic biological parameters of V. dahliae including colony diameter, sporulation, and pathogenicity were tested. The genetic variation characteristics were analyzed by AFLP molecular marker. The main results were shown as below.1. Two hundred and sixty one strains of plant pathogenic fungi preserved in mineral oil from 1951 to 1985 were recovered by PDA plate method. Results indicated that the survival percentage of plant pathogenic fungi decreased with the preserved time extension, and colony diameter and sporulation had no correlation with preserved years.2. Pathogenicity of V. dahliae showed that strain 45, 416, 193, 196, 164, and 165 among 19 testing strains preserved in mineral oil at room temperature for 17 to 52 years still had a certain pathogenicity to cotton. The strain 45 and 416 preserved over 50 years still had high pathogenicity. Up to now, they are the longest V. dahliae preserved in mineral oil at room temperature.3. The region of ITS (Internal transcribed spacer)1-5.8S-ITS2 sequences of 45 strains were amplified and sequenced using primer ITS1 and ITS4. The Basic Local Alignment Search Tool (BLAST) results showed that strain 3, 4, 5, 8, 13, 45, 164, 165, 181, 193, 196, 414, 416, 0-3, 0-9, 0-11, 0-12, 0-18, 0-21, 0-25, 1-4, 1-5, 1-9, 1-11, 3-4, 3-5, 3-6, 3-12, T-9, t13, V23, 1991, 2008, AY, JS, HT, and JY are V. dahliae, and strain 43, 55, 152, 286, 287, 288, 289, and 394 are V. nigrescens. One nucleotide of ITS sequence of strain 394 was different from that of other V. nigrescens.4. Fourty five strains were analyzed using amplified fragment length polymorphism (AFLP) technique. Results showed that these strains had rich polymorphism of AFLP molecular marker. The phylogenetic tree of SAHN was constructed using UPGMA method based on AFLP data. These strains were clustered into two groups. One was V. dahliae group, the other was V. nigrescens group. It was agreed with the result of ITS sequence. Cluster analysis revealed that there was certain correlation between V. dahliae pathogenicity and polymorphism of AFLP molecular markers.