Studies On Cultivation Of Hirsutella Huangshanensis, Anamorph Of Cordyceps Formosana

The growth curve, cultivation conditions for shake culture, plate culture and rice medium culture of RCEF0868, a strain of Hirsutella huangshanensis, anamorph of Cordyceps formosana, was studied in the present thesis. Based on above studies, methods of infection taking Tenebrio molitor instead of host insect, was investigated also.The growth curve of RCEF0868 was studied with mycelia weight as indicator. The result showed that the growth curve of shake culture and plate culture are almost the same.The growth curve of shake culture was as follows. The lag phase was from inoculation to day 15. The growth of mycelia in this period was in low level. The log phase was from day 15 to day 25, mycelia growing at the maximal rate, with the maximum weight reached 0.5378g/100mL on day 25. From day 25 to day 30, the growth ceased and the growth curve becoming horizontal, it is stationary phase. From day 30 on, the decline of mycelia weight indicate the death phase. There is slight different in the growth curve of plate culture with different indicators. By the indicator of the diameter of colony, the stationary phase was from day 25 to day 40, and the death phase began later. If the weight of mycelia was considered as indicator, the maximum weight would appear at day 30, then wet weight and dry weight reached 0.3604g/dish and 0.0702g/dish. At the same time, the moisture content reached 80.54%.The effect of nutrition conditions, such as carbon and nitrogen sources, inorganic salts, and culture conditions, such as temperature, pH, dissolved oxygen, humidity, inoculum amount and light, on growth and development of RCEF0868 was studied. The result of studies on single nutritional factor indicated that glucose, peptone, yeast extract points, MgSO₄ ? 7H2O, ZnSO₄, K2HPO₄, CaCl2 can promote the growth of mycelia. Glucose was considered as the best carbon source. The effect of sucrose was not good as that of glucose. Starch, maltose andα-lactose were not suitable carbon sources. The optimal concentration of glucose was 40g/L. If the concentration is low, the carbon source can not meet the need of growth, but yet, high glucose concentration will inhibit the growth. The result showed that suitable nitrogen source was essential. RCEF0868 grew well when treated with organic nitrogen, but it did not grow on the medium with inorganic nitrogen and CK. Peptone and yeast extract were considered as the best nitrogen sources, and the optimal concentration was 10g/L. The result also indicated that appropriate inorganic salt had promotion effect on growth. Eight inorganic salts were tested, but only MgSO₄?7H2O, ZnSO₄, K2HPO₄, CaCl2 were suitable at certain concentration. The result of orthogonal experiment showed that the order of effect factors is ZnSO₄> MgSO₄?7H2O>CaCl2> K2HPO₄. The variance analysis of orthogonal experimental design showed that all of these four factors have reached significant levels (p <0.01). On the basis of the above-mentioned results, the optimal nutrition conditions were glucose 40g/L, peptone 10g/L, yeast extract 10g/L, ZnSO₄ 5μg/L, MgSO₄?7H2O 20μg/L, CaCl2 5μg/L , K2HPO₄ 10μg/L.Culture conditions, such as temperature, pH, dissolved oxygen, humidity, inoculum amount, illumination, effected the growth and development of RCEF0868. It grew well at pH6.0⁷.0, and pH6.5 was the best. The appropriate temperature and rotate speed were 25℃and 160rpm. The suitable inoculum amount is 5mL, spore suspension with the concentration of 1×108 spores/mL.The growth and development was strongly influenced by illumination. Mycelia grew vigorous in the dark environment,and illumination stimulated the transition to propagation stage. Illumination by white light in a particular critical period preceded by a dark period induced the primordial. The critical period was identified to be 20d after inoculation. The culture of rice medium was studied, in reference with the result above. The result showed that the suitable ratio of medium and water was 1∶0.5, by the weight of rice was 5g. The appropriate inoculum amount is 5mL, spore suspension with the concentration of 1×108 spores/mL.The comparison of three infection methods, dipping with spore suspension, dipping with mycelia suspension, touch with the colony, Tenebrio molitor as replaced host, was studied. The result suggested that dipping with spore suspension and dipping with mycelia suspension leaded to a low mortality of 8.33%and 15%. The method of touch with the colony got a better performance, and the mortality reached 18.33%.