Abstract
Verticillium dahliae is a causal agent of vascular wilt smoke-tree Cotinus coggygrial. The genetic transformation of V. dahliae has been carried out by Agrobacterium and polyethylene glycol-mediated transformation. Due to technical simplicity and efficiency, PEG-mediated transformation is the most commonly used method for genetic transformation in filamentous fungi. However, polyethylene glycol (PEG)-mediated transformation is still deficient in V. dahliae. In this study, we studied the main factors affecting the release of protoplasts from mycelia of V. dahliae such as lytic enzymes, enzymolysis buffers and incubation time. The optimal combination for protoplast preparation from smoke-tree wilt fungus V. dahliae can release 2.8 × 106 protoplasts/ml from fresh mycelia of smoke-tree wilt fungus. The majority of spheroplast contained nuclei and well regenerated. After PEG mediated transformation, green fluorescent protein (GFP) expressing V. dahliae strains were obtained. Therefore, this study provides a protocol suitable for protoplast preparation and transformation of smoke-tree wilt fungusV. dahliae. Key words: Verticillium dahliae, smoke-tree wilt, protoplast preparation, polyethylene glycol (PEG) transformation
Highlights
Verticillium dahliae is a soil-borne vascular wilt fungus which infects over 200 plant species and causes annual crop losses of billions of dollars
We studied the main factors affecting the release of protoplasts from mycelia of V. dahliae such as lytic enzymes, enzymolysis buffers and incubation time
Among seven different treatments tested, the results showed that 20 ml of 0.7 M NaCl with mixed enzymes (0.1 g lysing enzyme and 0.2 g driselase) was capable of releasing the greatest number of protoplasts from mycelia of V. dahliae strain XS 11 after incubation at 30°C for 6 h; a significantly higher number of protoplasts (2.8 × 106 /ml) than in the other treatments (Table 1)
Summary
Verticillium dahliae is a soil-borne vascular wilt fungus which infects over 200 plant species and causes annual crop losses of billions of dollars. V. dahliae enters and colonizes the plant xylem system, disrupting water transport, and causing the characteristic symptoms of wilting, and often vascular discoloration (Klosterman et al, 2009; Klosterman et al, 2011). In Beijing Fragrant Hills Park, the smoke-tree, Cotinus coggygria, is one of the most important cultivated plant species for the landscape ecology, and is the main component of the red leaf scenery during autumn. It was initially infected by V. dahliae during the 1990s, and the disease soon spread widely through the region. The main symptoms are stunted stem growth, early leaf senescence, and severe mortality, with seriously detrimental effects on the red leaf scenery (Wang et al, 2008)
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