Effect Of Mineral Elements On Phenotypic Diveristy, Fruit-body Yield, Anamorph Diveristy And Activity Of Related Enzymes Of Colony Types Of Cordyceps Militaris In Subculturing

L20, a good strain, was subcultured on different mineral elements media for twelve times with orthogonal experimental method. There were seven colony types of L20 after subcultured. To elucidate the relationship among mineral elements, phenotypic variation of colonies, anamorph diversity, activity of related Enzymes and fruit-body yield of different colony types of Cordyceps militaris in Subculturing; effects of mineral elements on the colonial morphology diversity, maintainning generations, growth rate of mycelia, dry weight of hypha ball, frult-body yield, section rate, anamorph diversity,sporulation,activity of Esterase, superoxide dismutase(SOD)and CMCase of different colony types of Cordyceps militaris in subculturing were investigated. Main results were as follow:1. The results showed that there were seven colony types of L20 after subcultured, among which typeⅠ,Ⅱ,Ⅲand V had ability to form fruit-body; however, typeⅣ,ⅥandⅦcouldn't form fruit-body. Of these,typeⅠperformed the highest yield of fruit-body, and typeⅡthe lowest, with typeⅢstanding in between,while all the fruit-body produced by typeⅤwere abnormal. With increasing generation of subculture, the fruit-body of typeⅠ,Ⅱ,Ⅲfirst increased and then decreased, and the yield of fruit-body more higher among mid-generations. The formulation of mineral elements, which kept typeⅠhigh yield and maintained more generations was K2Mg2Ca3Mn3Zn2, of which Mn and K were able to produce high yield of fruit-body; K, Mg and Ca could maintain more generations to produce high yield. The medium contained K3Mg2Ca4Mn3Zn2could promote typeⅡproduce more fruit-body, Zn and Ca had the most significant effects but Mn had negative effect.2. Distribution patterns of colony types in different media were as follow:typeⅠandⅢwhich had higher yield of fruit-body mainly distributed in 1-10 generations; lower yield colony typeⅡwas distributed in 1-4 generations; typeⅤwhich formed abnormal fruit-body andⅣ,ⅥandⅦwhich have no ability of forming fruiting-body mainly appeared after 4 generations.3. Different colony types had different maintaining generations. typeⅠmaintained most generations and typeⅡthe lowest, with typeⅢstanding in between.Different colony types had different maintaining generations in different mineral elements media.The formulation of mineral elements, which kept typeⅠmaintained more generations was K3Mg1Ca2Mn1Zn3/4, of which K, Ca and Zn had the most significant effects,but Mg and Mn had negative effects. TypeⅡmaintained more generations in medium which contained K2Mg4Ca4 Mn2Zn4, and TypeⅢmaintained more generations in medium which contained K4Mg2Ca2 Mn4Zn3, These five mineral elements could all extend maintaining generations of typeⅡandⅢ.4. With generation of subculture increasing, the growth rate of mycelia of different colony types proformed leggy. The growth rate of mycelia of different colony types were as follow:typeⅤ>typeⅣ>typeⅦ> typeⅥ> typeⅢ> typeⅡ> typeⅠ. Its showed the colony which had slowest growth rate could produce most fruit-body. The best formulation which suppressed mycelia leggy of type I was K3Mg2Ca2Mn3Zn2, Mn and Zn had the most significant effects. The best formulation which suppressed mycelia leggy of typeⅡwas K2 Mg4 Ca3 Mn1 Zn4, Zn and K had the most significant effects but Mn had negative effect.5. With generation of subculture increasing, dry weight of hypha ball of different colony types were increasing. The dry weight of hypha ball of different colony types were as follow: typeⅡ>typeⅠ>typeⅣ>typeⅦ>typeⅥ>typeⅢ>typeⅤ. The best formulation which suppressed dry weight of hypha ball of typeⅠwas K3Mg3Ca3Mn2Zn2, Ca and Mn had the most significant effects. The best formulation which suppressed dry weight of hypha ball of typeⅡwas K2Mg2Ca3Mn4Zn3, K, Mg and Zn had the most significant effect.6. The investigation results of section showed with generation of subculture increasing, section rate was increasing. The section rate of colonies which could happed sector were as follow:typeⅠtypeⅢ>typeⅡ> typeⅤ >typeⅣ>typeⅦ>typeⅥ. The medium contained K.2Mg3Ca3Mn3Zn2 could enhanced the activity of Esterase of typeⅠ, Ca,K and Mn had the most significant effects. The medium contained K2Mg3Ca4Mn4Zn4 could enhanced the activity of Esterase of typeⅡ, K,Ca and Zn had the most significant effects.10. The activity of SOD much higher among mid-generations of these colony types. Activity of SOD of different colony types as follow:typeⅠ>typeⅢ>typeⅡ>typeⅤ> typeⅣ>typeⅦ>typeⅥ, typeⅥhad highest degree of caducity. The medium contained K.3Mg3Ca3Mn3 Zn2 could enhanced the activity of SOD of typeⅠ, Zn and Ca had the most significant effects. The best formulation which hept the highest activity of SOD of typeⅡwas K3Mg3Ca2Mn3Zn2, Zn,Ca and K had the most significant effects.The results showed Zn and Ca could enhance the ability of anti-aging of strain.11. Cellulose was a multienzyme cluster, activities of different kinds of cellulases much higher among mid-generations of these colony types. Activity of CMCase and 4-β-D-glucanase (cellobiohydrolase, CBH) of different colony types as follow:typeⅠ> typeⅢ> typeⅡ> typeⅤ> typeⅣ> typeⅦ> typeⅥ, it showed typeⅠhad highest activity of Cellulose. L20 had higher ability to produce CMCase, and 4-β-D-glucanase the lowest, with FPA standing in between. The best formulation which hept the highest activity of CMCase of typeⅠwas K3Mg3Ca2Mn3Zn2, K had the most significant effect. TypeⅡhad highest activity of CMCase in medium which contained K4Mg3Ca3Mn4Zn2, Mn, K and Ca had the most significant effects. The medium contained K2Mg2Ca2Mn3Zn2 could enhance 4-β-D-glucanase activity of typeⅠ, of which K and Ca had the most significant effects; and the medium contained K4Mg2Ca4Mn3Zn2 could enhance 4-β-D-glucanase activity of typeⅡ, of which Mg had the most significant effect. Type I had highest FPA activity in medium which contained K4Mg2Ca4Mn3Zn2, Mg had the most significant effect; and Type II had FPA activity in medium which contained K4Mg2Ca3Mn2Zn4, Mg also had the most significant effect.12.Correlation analysis revealed sporulation, Esterase activity, SOD activity, CMCase activity,4-β-D-glucanase activity and FPA activity had extremely significant correlation (P<0.01) to fruit-body yield of TypeⅠ,of which SOD had the most correlation. The result showed TypeⅠperformed the highest activity of these enzymes, stronger metabolism, stronger ability of anti-aging, most stable property and highest yield of fruit-body. Growth rate, dry weight of hypha ball and section had negative correlation to TypeⅠfruit-body yield, but relevance was not significant. The results showed the mycelia of typeⅠnot easy to leggy, so it had small effect on TypeⅠfruit-body yield.Overall, TypeⅠwas the best colony type, the features of TypeⅠas follow:the colony color preformed saffron yellow, texture like dense floss with concentric circle, Aerial hyphae in intermediate level, growth rate was slower, the mycelia biomass was little in liquid media, colony could happed sector,but the section was smaller than other colonys which could also happed sector in subculturing,and Type I performed the highest fruit-body yield, maintained more generations, had highest activities of Enzymes, stronger metabolism, stronger ability to produce Enzymes and had stronger ability of anti-aging; anamorph feature was both has two kinds of spore-bearing structures(Paecilomyces-type and Cephalosporium sp-type). The studies also showed had TypeⅠhigher fruit-body yield and stronger metabolism among mid-generations. we can domesticate more generations in breeding for gurantee highyield and good stability. The formulation of mineral elements, which kept typeⅠhigh yield and maintained more generations was K3Mg2Ca4Mn3Zn2. K had most positive effect on many aspects, followed Ca and Mn, Zn and Mg proformed a minor role.