Monitoring Spatial Distribution of Nonpathogenic Fusarium Oxysporum in the Rhizosphere and Roots of Banana Using Β-D-Glucuronidase and Green Fluorescent Protein
Wong , Pui See (2005) Monitoring Spatial Distribution of Nonpathogenic Fusarium Oxysporum in the Rhizosphere and Roots of Banana Using Β-D-Glucuronidase and Green Fluorescent Protein. Masters thesis, Universiti Putra Malaysia.
This study was conducted to monitor the spatial distribution of nonpathogenic isolate of Fusarium oxysporum (Fo4), using a detectable marker. Fo4 was transformed by GUS gene fusion system (Escherichia coli β-D-glucuronidase gene) and GFP (Green Fluorescent Proteins). The GUS was detected by 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc). The GFP activity was detected under a fluorescence microscope. There was no different in the cultural and morphological characteristics between the transformed and non-transformed Fo4. The antagonistic activity against Fusarium oxysporum f. sp. cubense Race 4 (FocR4) was maintained at values within the range of 56.98–41.56% based on the Percentage Inhibition of Radial Growth (PIRG) in vitro. DNA polymorphism (RAPD) screening by the primers OPC-15 and OPC-11, confirmed that no mutation had occurred after the transformation and, band GUS-F vs. GUS-R and 5-GFP vs. 3-GFP showed that transformed Fo4 still carried the GUS and GFP gene after five consecutive conidiation/ generation cycles in culture. The frequency of re-isolation on PCNB medium amended with 40 µg/ml X-Gluc and 200 µg/ml hygromycin B was highest (45.48 x 103 cfu/g roots) in the rhizosphere of banana plantlet at day 24, and remained to stabilize at between 37.24 x 103 cfu/g roots to 34.52 x 103 cfu/g roots until the end of sampling at day 36. The transformed Fo4 can be detected inside the root tissues even 4 days after inoculation although the colony forming units (cfu) (1 cfu/g roots) was substantially lower than that detected on the root surface (11.76 x103 cfu/g roots), suggesting that they were colonizing the root and living on the root exudates. The population of transformed Fo4 in the non-rhizosphere soil initially fell sharply with the time of sampling, but thereafter remained stable (4.38-18.00 x 104 cfu/g soil) until 36 days of sampling. Effect of transformed and non-transformed Fo4 on plant growth and development of Fusarium wilt was conducted using 10 week-old tissue cultured nursery banana seedlings cv. Berangan. The failure to produce Fusarium wilt symptom confirmed this strain was nonpathogenic to banana seedlings. The seedlings inoculated with FocR4 were pathogenic to cv. Berangan with 100% disease severity. However, the seedlings which had been inoculated with either transformed or non-transformed Fo4 before challenged with FocR4 did not show significant differences (P>0.05) in the disease severity, confirmed the stability of the transformed GUS activity and GFP. This study suggests the possibility of using the GUS and GFP genes as visible detectable reporter genes for direct monitoring of the spatial distribution of a potential antagonist in the rhizosphere and roots of banana.
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