Study On The Optimization Of Cultural Conditions For Submerged Sporulation And Infectious Process Of Cordyceps Militaris

Cordyceps militaris (L.) Link. is an edible and medicinal fungus bearing various pharmacological activities. It is also an important entomopathogen which can parasitize in many kinds of insects belonging to Lepidoptera, Coleoptera and Diptera. Great effort has been devoted to explore its medicinal efficacy while little attention paid to its biocontrol potential. In the present study, morphological observation was made to clarify the confusions of the diagnostic characteristics and taxonomical status of anamorph of C. militaris. Statistical experiment designs were employed to optimize the culture conditions for submerged sporulation of C. militaris to facilitate its further exploration as a biopesticide. To better understand the interaction of fungi with their host insects and for better utilization and protection of Cordyceps (Fr.) Link, the infectious mechanism of C. militaris on silkworm chrysalis was also studied.The aerial conidiation structure of anamorph of C. militaris was found to be pleomorphic through morphological study. The phialides, aculeate or flask-shaped, grew singly or in whorls while the globose or pyriform-shaped conidia aggregated in imbricate chains or misshaped spore heads.Sequential observation on the ontogeny of submerged spores of C. militaris revealed that there were three different modes of sporulation, including typical conidiogenesis via phialides, microcycle sporulation and multiplication by fission.Nutritional requirements for submerged spore production of C. militaris were studied by using one-factor-at-a-time method. Galactose, soybean peptone, vitamine B12 were identified as the best carbon, nitrogen and growth factor sources for the optimum viable spore production respectively. Then a 2^7-3 fractional factorial design (FFD) was employed to evaluate the effects of seven nutritional and environmental factors, i.e. carbon to nitrogen (C:N) ratio, vitamine B12, flask capacity, pH value, inoculum size, temperature and rotary speed, on submerged spore production of C. militaris. C:N ratio and flask capacity were found to be the most significant factors, whereas other five factors were not important within the levels tested. The method of steepest ascent was then used to approach the proximity of optimum. In the fourth step, the optimal levels of C:N ratio and flask capacity were determined by a central composite rotatable design (CCRD) and response surface analysis. The optimized submerged culture conditions were found to be: C:N ratio 1．18 and flask capacity 35/250 mL. Under the optimal culture conditons, the viable spore yield of 1．88×10⁸ spores mL^-1 was obtained 132 h after inoculation, which was in coincidence with the predicted maximal value of 1．74×10⁸ spores mL^-1 .Further study was carried out to explore the infectious mechanism of C. militaris on its host by investigate the effects of various chrysalis pre-treatments, inocula types and inoculation methods. Injecting a suspension of submergd spores of C. militaris was found to be most effective method of successful infection.