Methods for Identification and Detection of Fusarium oxysporum f. sp. gladioli , the Causal Agent of Gladiolus Fusarium Wilt and It’s Dissemination

• Main Authors: Yu-Chu Chen, 陳昱初
• Other Authors: W. H. Hsieh
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/34237599353768569360

博士 === 國立中興大學 === 植物病理學系所 === 94 === Gladiolus Fusarium wilt , caused by Fusarium oxysporum f. sp. gladioli, is the most of destructive soil-borne disease during it’s growing period. Isolation and the identification of the pathogen was not only laborious but also time-consuming. It is necessary to develope an efficient technique to estimate the population of F. oxysporum f.sp. gladioli ,the gladiolus Fusarium wilt pathogen, in natural soil and diseased plants, which is applicable to research on ecology of the pathogen. The objective of this study was to develop a selective medium for isolation and detection of F. oxysporum f.sp. gladioli. The selective medium (pH2.0) consisted K2HPO41g/L, KCL 0.5g/L, MgSO4·7H2O 0.5g/L, Fe-Na-EDTA 0.05g/L, L-Asperagine 2g/L, D-galactose 20 g /L, PCNB 1g/L, Oxgall 0.5g/L, Streptomycin sulfate 300ppm, Benomyl 1000 ppm, agar 20g, distilled water 1L. Test result proved that F. oxysporum f.sp. lilii , F. oxysporum f.sp. luffae , F. oxysporum f.sp. cucumerinum , F. oxysporum f.sp. tracheiphilum , F. oxysporum f.sp. monodricae , F. oxysporum f.sp. melonis , F. oxysporum f.sp. cubensei and non-pathogenic F. oxysporum were not able to grow on the selective medium plate. This medium was more sensitive than those of Nash-PCNB and Komada for distinguishing F. oxysporum f.sp. gladioli from the other Fusarium species. The recovery rates of F. oxysporum f.sp. gladioli (FOG051) from the artificially infested soil was 96%. RAPD was used first to screen the total DNA of pathogenic and non- pathogenic Fusarium oxysporum isolates using 100 random primers. The primer OPAW-06 (5''-TTTGGGCCCC-3'') was selected, and the specific DNA segment (890bp) was amplified in the total DNA of pathogenic isolates present. The specific DNA segment amplified was recovered, cloned, and sequenced. The specific primer set FOG-W6L-001F/FOG-W6L-006R was designed and assembled according to the nucleotide sequence of the specific DNA segment. The detection of F. oxysporum f.sp. gladioli from mycelium can be conducted using the FOG-W6L-001F/FOG-W6L-006R specific primer. The results showed that the specific primer set developed can successfully detect F. oxysporum f.sp. gladioli in pure culture. In Kaohsiung-Pingtung area disease occurrence and distribution in monoculture fields had been studied from March 2002 to March 2003. According to Lloyd,s index analysis, field distribution of the disease has a spatial aggregation that matched a grid pattern plotted by disease severity. The results showed that the disease was soil-borne. In addition to being soil-borne, infected gladiolus corms causing disease in field were also found in this study and were correlated to disease severity based on the data from Dunggang and Wandan in Pingtung County. This evidence proved that F. oxysporum f. sp. gladioli was transmitted through either infested soil or infected gladiolus corms.