Improvement Of Fusarium Oxysporum FO47 By Protoplast Fusion

The effective wild strains were isolated from nature, but the strains showed non-stable control effects and poor resistance to fungicides. So the wild strains need to be improved to enhance the ability of control diseases and adaptability to environment. Non-pathogenic Fusarium oxysporum FO47 showed inhibition against Fusarium oxysporum and could promote the growth of plants, but sensitivity to carbendazim. Actinomycetes 153 showed more resistance to fungicides and could colonize in the tissues of plants. In order to improve the characters of FO47, the excellent characters of non-pathogenic Fusarium oxysporum Fo47 and actinomycetes 153 were tried to integrate into a recombinant by protoplast fusion. The study mostly included content as follows:First, the best situations of protoplast formation and regeneration of the parents were determined by experiments. The strain 153 was cultured in two-step method. Firstly, strain 153 was inoculated in the mycelium culture fluid containing 20% sucrose and cultured for 48h under the condition of 25℃and 150rpm. Then, strain 153 was transferred into mycelium culture fluid containing 30% sucrose and 0.5% glycin under the same condition for 24h. In order to get great number and high quality protoplast, the mycelium of strain 153 was treated by 7.5mg/mL lysozyme and 7.5mg/mL snailase (1:1) at 35℃for 120min, while the spores of strain FO47 were treated by 10mg/mL snailase and 5mg/mL lywallzyme (1:1) at 35℃for 120min. The protoplasts of the parents FO47 and 153 were inactivated by heat at 55℃for 15min and 20min, respectively. Both protoplasts were inactivated by UV for 5min and 15min, respectively. The death rate of protoplasts of strains FO47 and 153 treated by the ultrasound of 40 kHz and 200W in the process of 10min and 15min were 99%, respectively.Second, based on single or bi-parent inactivated, protoplast fusion was carried out by 35% PEG6000 at 35℃for 10min. Fourteen recombinant strains whose growth rates were higher than that of strain FO47, were selected based on morphological and cultural characteristics.Three, three recombinant strains were further selected according to greenhouse pot experiments and resistance to carbendazim. Strains F1-35 and F1-38 could promote the growth of the seedlings of watermelon. The content of gibberellin produced by strains F1-35 and F1-38 were higher than that of strain FO47. Strains F1-1and F1-35 could colonize in the tissues of cotton, cucumber and watermelon. Strain F1-4 showed more resistance to carbendazim.Protoplast fusion was carried out between fungi and actinomycetes by inactivation and mutation in order to improve the characters of strain FO47. Also excellent recombinant strains were got to be used in the biocontrol of plant disease.