| Banana Fusarium wilt disease, caused by the filamentous fungus Fusarium oxysporum f. sp. Cubense (Foc),is one of the most devastating diseases of banana, distributing on many banana-growing areas in the world.The disease is also one of the most devastating diseases of banana in China,and there are diseases caused by Foc race 4 in Hainan,Guangdong,Fujian.For most banana-growing areas,banana Fusarium wilt management relies on the frequent application of new resistant banana cultivars. However, the management of banana Fusarium wilt through the use of disease-resistant cultivars has become difficult because of the highly variable nature of the pathogen as well as favorable environmental conditions for infection during the crop season.Therefore, genetic diversity in the Foc population was studied to facilitate the banana breeding for durable resistance and the disease integrated management.The PEG-mediated transformation system of Foc was established for aiding to gene clone,studing expressing regulation theory of gene,gene function. And transformation of the green fluorescent protein gene into Foc was also studied for facilitating future infection studies with this pathogen on banana.In China,we firstly reported the biological characteristics of race 4 strain Foc-37,and comparatively studied the biological characteristics of race 1 strain Foc-2 and Foc-37.The optimum temperature for mycelial growth was 25℃.The optimum pH was 8,and Foc-37 grew better than Foc-2 at acidity condition,but less at pH10-11. The fungus could use 9 kinds of carbon,and grew worst at sorbose carbon; Foc-37 grew best at dextrose carbon,and Foc-2 grew best at maltose carbon.The optimum temperature for conidial germination was 28℃;germination rate curve of Foc-37 was 2-apex type,germination rate reducede obviously at 24℃,and the germination rate of Foc-37 was higher than that of Foc-2 at same temperature .The optimum pH for conidial germination was 5,and the germination rate of Foc-37 was higher than that of Foc-2 at same pH .We firstly reported there was Foc race 4 in Hainan,China,providing theory base for government adopting efficient management.The virulence of strain Foc-20 from Guangdong was the most among the tested strains,so Foc-20 could be used to infection,when screening resistant race 4 cultivars.Primer OPM-15 could be used to identify race 1 and 4.RAPD analysis could differentiate geographic origin of isolates of race 1 and 4,approximately distinguish isolates of race 1 and 4,and distinguish pathogenicity of isolates of race 4.Vegetative compatibility groups of fourteen single-spore Foc isolats in Hainan were firstly tested by the technology of nitrate reductase deficiency. All of 14 isolates produced 104 chlorate-resistant sectors on KPS media and 67 nitrate non- utilizing mutants (nit mutant), which were recovered by transferring the chlorate-resistant sectors to the MM media containing nitrate as single nitrogen source. Among the 67 nit mutants, 58 nit mutants were nit l, 6 nit mutants were nit M, and 3 nit mutants were nit 3/nit 8. Complementation of vegetative compatibility tests were carried out on MM media by pairing the nit mutants from the same strain and from different strains. 14 strains were assigned to 2 vegetative compatibility groups, 7 strains ( race 1) from Fenjiao in VCG 1, 7 strains (race 4) from Xiangjiao in VCG 2,showing vegetative compatibility groups of Foc isolats in Hainan had some relations with host origin and race.The PEG-mediated transformation system of Foc was established. Conidial suspensions were inoculated into potato-dextrose liquid media and placed on a shaker at 180r/min,28℃for 24 h.Cultures were filtered with 4-layer gauze and then washed with 0.8 mo1/L NaCl solution.5 mL 10mg/mL enzyme solution and 250 mg wet mycelium were added to 50 mL sterile triangle flask,and incubated at 80r/min,30℃for 3.5 h. Cultures were filtered with 3-layer rubbing lens paper,and the filtrate was transferred to 15 mL sterile needle-bottom centrifuge tubes (the same below),and centrifuged for 10min at 4000 r/min,4℃(the same below).The deposition was gently suspended with 3mL 1 mol/L MgSO4 solution.After centrifuge,the same volume sterile distilled water was slowly added to the supernatant. The spheroplast solution was centrifuged,and the deposition was washed with sorbito1 solution (STC) (1.2 mol/L sorbito1;10 m mol/L Tris-HCl (pH 7.5); 10 m mo1/L CaCl2) two times.After centrifuge,The spheroplasts (at 2×107-3×107 cells/mL) were finally resuspended in STC solution.For the transformation, spheroplasts (2×107-3×107/mL in 1 mL) were mixed with 10μg plasmid,25μL 2×STC(2mol/L sorbito1;100 m mol/L Tris-HCl (pH 8.0);100mmo1/L CaCl2) and 25μL 60% PEG 4000 in a 15 mL tube,then incubated at room temperature for 20 min. An additional 1.2 mL of 60% PEG 4000 was then added, mixed gently and the mixture incubated for exactly 5 min at room temperature.4 mL STC was added and mixed.The mixture was centrifuged at 3000r/ min for l0min,and the deposition was suspended with 2 mL STC.200μL protoplast suspension was plated on PDA regeneration medium containing 0.8 mo1/L sucrose.The plates were incubated right side up at 28℃.After12 h, each plate was overlaid with 10mL of 1 % water agar containing gromycin-B at a concentration of 150μg/mL.Plates were incubated right side up at 28℃.Transformed isolates grew through the overlay in the presence of gromycin-B within 6-7 days.The transformed isolates did not differ markedly from the wild type isolates in growth and morphological characteristics in vitro. Fluorescence microscopy showed expression of the green fluorescent protein (GFP) in fungal structures after transferring 9 times. The presence of the green fluorescent protein DNA in the fungal cells was confirmed by PCR using a GFP-specific primer pair. Pathogenicity tests showed that the transformation process did not alter pathogenicity of Foc isolates.The transformed fungus was re-isolated from artificially inoculated plants.Transformants of Foc will facilitate future infection studies with this pathogen on banana. |
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