Study On The Biological Characteristics Of The Degenerated Strain Of Cordyceps Militaris

The degradation of strain seriously affects the stability of intensive production and the healthy development of Cordyceps militaris industry.The Cordyceps militaris strain would degrade after several times of subculture.The degeneration is usually manifested as the reduce ability of scavenging reactive oxygen species.In order to study the relationship between the degradation characterization and degradation degree with the active oxygen metabolism.In this experiment,the normal strain W5,half degradation strains W5-1 and degradation strains W5-2 isolated from the Cordyceps militaris strains of wheat storage were used as the test materials.The morphology,physiological and biochemical characteristics of degradation strains has carried on the exploration.We observed the differences in microstructure and macro characterization,and determinated the activity change of eight kinds of antioxidant enzymes related to the metabolism of reactive oxygen species(ROS).The results showed that(1)By cloning and doing the comparative analysis of the ITS sequences of Cordyceps militaris W5,W5-1 and W5-2.Founding that the similarity of ITS sequences of the three strains reached 98.66%,with high homology,.which indicating that the test strains were isolated from the same strain,and the comparison of the three strains has the biological significance.(2)The growth and development rate of degraded Cordyceps militaris colonies changed significantly in time and space.The colonies of semi-degraded and degraded strains were pale yellow with white edges.The air-born mycelia were vigorous and long,and obvious rhythm rings could not be seen.The growth rate of the degraded strain was significantly lower than that the normal strain and the semi-degraded strain in the early stage of culture.While in the late stage,the growth rate of the degraded strain and the normal strain was significantly higher than that the semi-degraded strain.(3)In terms of macro characterization,the semi-degraded and degraded strains showed slow growth,longer culture cycle,lower yield and quality of friuts,and the uniformity and deformity had decreased.(4)In terms of microscopic characterization,the subcellular structure and organelles of the strain changed significantly with the aggravation of degradation.In terms of hypha appearance structure,the normal hyphae was straight and full,the surface of cell wall was smooth.The half part hyphae of the semi-degraded strain were full,but we also can found the slender and contorted hyphae.The cell wall of semi-degraded strain was thinning,and the surface was rough.And there wer some concave pit on the surface of the cell wall.A large number of hyphae in degenerated strains were severely contorted,and the diaphragm is not evenly distributed.The cell wall were constricted and shrivelled of degradation strains.Inside of the cell,the cytoplasm of the normal strain was filled,and there were a lot of nucleis and little mitochondrias in the cell.In terms of the cells with different degrees of degeneration,part of the cell membrane margin were irregular,and there were many large vacuoles in the cell,and even formed a large number of cavities by mutual fusion.There were fewer nucleis and a lot of small mitochondrias.And the accumulation of reactive oxygen species at hypha tip was obviously increased.(5)The activity of 8 antioxidant enzymes(SOD,CAT,POD,APX,GPX,GR,DHAR and MDHAR)in the anti-oxidase system was measured,and it was found that the enzyme activity of SOD,GR and DHAR in the normal strain was extremely significantly greater than that of the semi-degraded and degraded strain,while the activity of MDHAR in the normal strain was significantly greater than that of the degraded strain.GR,DHAR and MDHAR are the key enzymes in the ASA-GSH cycle,so it is speculated that the degradation of Cordyceps militaris is related to the recycling of ascorbate and glutathione,which provides a viewpoint for the research on the degradation of Cordyceps militaris.(6)On the correlation analysis of SOD,GR,DHAR,MDHAR,hypha cell wall thickness and diameter,it was found that the GR activity and the rest of the five variables have the significant or extremely significant positive correlation,the activity of SOD and of the four variables had the significantly or extremely significantly positively positive correlation in addition to the cell wall thickness.So it can be thinking that the activity of GR and SOD could represent the degradation degree of hyphae in a certain extent,which suggestting as the appraisal index of the degradation of the Cordyceps militaris strains.