Ferment Condition Optimization And Isolation, Gene Cloning, Characterization Of A Pigment Mutant Of Lysobacter Enzymogenes Strain OH11

The control of plant diseases,caused by fungi,bacteria,and nematodes is a key problem in agricultural production.Till now,many plant diseases were controlled by use of chemical pesticides,which resulted in environmental popullation and doing harm to human beings.Recently,biological control of plant diseases is more and more concerned because of its safety for environment and human beings.In addition, biological control is effective to control some plant diseases for a long time.Members of the genus of Lysobacter are typically found in soil and water habits and are characterized by a high G+C content,gliding motility,and the ability to lyse other microbes,including other bacteria,fungi,and nematodes.One member of this genus,Lysobacter enzymogenes,characterized by the ability to produce extracellular lytic enzymes,including chitinases,β-1,3-glucanases,and proteases,has been described as potential biological control agents for plant diseases.In this study, fermentation condition optimization and characterization of a pigment mutant of Lysobacter enzymogenes strain OH11 was performed.The fermentation of Lysobacter enzymogenes strain OH11 was investigated through single-factor test,the main factors include:temperature,rotate speed.The optimal fermentation conditions are:temperature 30℃,rotate speed 150 r/min.The orthogonal experiment was used to optimize the medium,and th number of strain OH11 were improved from 8.5×10⁹ CFU·mL^-1 to 1.2×10⁹ CFU·mL^-1 under optimal condition after 72 h.The fermentation condition of OH11,such as volume, fermentation time,inoculum's size and pH value before sterilized were tested by orthogonal experiment.The optimum fermentation conditions are as follows:volume 40 ml/250 ml,fermentation time 72 h,inoculum size 10⁸ CFU,pH7.5 before sterilized, and studied the livability of Lysobacter enzymogenes strain OH11 under different conditions.The mutant library of Lysobacter enzymogenes strain OH11 was successfully constructed by mating mariner transposon into strain OH11.One pigment mutant OH11-1,which could produce dark-brown pigment on LB plate and in LB broth,was selected by the change of colony morphology.The transposon insertion site was identified as hmgA gene by sub-clone strategy.A hmgA disruption mutant OH11-3, which had the same phenotype as strain OH11-1,was constructed by a plasmid insertion inactivated protocol.The brown-pigment was identified as homogentisate by HPLC method,which was in accordance with that of sequence analysis.The results of detection of lytic enzymes,antifungal activity and greenhouse experiment indicated that:(â…°) strain OH11-2 had decreased ability to produce protease andβ-1, 3-glucanases,but retained that to produce chitinase and cellulase;(â…±) strain OH11-2 retained the antifungal ability against Rhizoctonia solani compared to wild type strain OH11;(â…²) strain OH11-2 displayed significantly decreased effect on biocontrol tomato wilt disease in green house compared to wild type strain OH11.