Morphological Characteristics Of Harposporium And Culture Optimization Of High Protease Producing Strain

Objective: To study the morphological characteristics of 15 Harposporium strains,screen for the strain of high protease and nematocidal activity and optimize their fermentation conditions and growth conditions,laying a foundation for further study on the molecular mechanism of nematocidal activity of Harposporium,and providing a reference to biological control of plant parasitic nematodes.Methods: 15 Harposporium were cultured,involving 1 strain of Harposporium sinense,1 strain of Harposporium oxycoracum,1 strain of Harposporium multisporum,1 strain of Harposporium thaumasium,2 strains of Harposporium lilliputanum,2 strains of Harposporium anguillulae,3 strains of Harposporium helicoides and 4 strains of unknown Harposporium sp.,in addition,some strains were described in detail.To screening a strain of high protease activity,the protease activity of all strain was determined by Folin-Ciocalteu method.Then fatality rate of nematodes was determined,the highly active nematocidal strain was screened.Comparing the protease activity of strains,the enzyme producing medium of high protease activity and nematocidal activity strain H12 was optimized,including single factor experiment and orthogonal design on the mass concentration of glucose,gelatin,peptone and yeast extract.The fermentation conditions were optimized by single factor experiment,including pH,temperature,rotation speed and inoculation amount.To screen carbon source and added nitrogen source in H12 potato glucose liquid medium is by comparing the dry weight of mycelia.The composition of medium and its culture conditions were optimized by the single factor test including glucose,yeast extract,pH,temperature,rotation speed,inoculation amount and the orthogonal design including glucose,yeast extract,pH,inoculation amount.Results: Colony culture of the Harposporium showed that the colony grew slowly,and the diameter of the colony increased by about 2 cm in 30 days.It could produce pigments with different colors.The hyphae of Harposporium are colorless,branched and separated.Conidiophore is colorless with the phialide on it.Phialide is spherical,subspherical or long elliptic with one or more pedicels on it.Conidia vary in shape,such as arch,helix and crescent.High yield protease activity and nematocidal activity strain H12 were screened from 15 strains of Harposporium.The highest protease activity was found on the 5th day of fermentation.The optimum medium composition was 8 g/L glucose,8 g/L gelatin,8 g/L peptone,4 g/L yeast extract.The optimum enzyme producing conditions were inoculation volume 2 fungus pieces with 5 mm diameters,temperature 20 ?,pH 7.0 and rotational speed 260 r/min.The optimum medium of strain H12 growth was based on liquid culture medium that the glucose concentration was increased to 40 g/L and the yeast extract concentration was 25 g/L.The optimum culture conditions were inoculation volume 4 fungus pieces with 5 mm diameters,temperature 30 ?,pH 9.0 and rotational speed 180 r/min.The protease activity of strain H12 increased to 216.42 U/mL,2.19 times higher than that before optimization(67.84 U/mL),and the dry weight of strain H12 was 1624.7 mg,3.63 times higher than that before optimization(351.2 mg).Conclusion: The morphology of Harposporium is diverse.Strain H12 has high activity of protease production and nematocidal activity.The optimized enzyme producing medium and enzyme producing conditions increase the protease activity of strain H12.The optimized growth medium and culture conditions accelerate the growth rate of strain H12.