Biological Characteristics And Possible Mechanism Of Dormancy On Aphid-pathogenic Fungi Conidiobolus Thromboides(Entomophthorales)

Conidiobolus thromboides is significant pathogenic fungi, a promising biocontrol agent for aphids. It could cause the epizootic for controlling the aphid populations through contacting and invading processing. In this paper, C. thromboides isolate Ct10201from the continent of South Africa, was studied for exploring the primary dormancy-related biological characteristic for further field application.Firstly, a rapid, novel and effective Photoshop-based method was developed to measure the fungi colony growth rate. Based on this novel area-calculating technique, several basic biological characteristics of this isolate were illustrated. Optimum growth temperature region of Ct10201was among25℃to30℃. As for optimal pH, growth rate in neutral pH (pH7and pH8) was better than that of other pHs. Although light was not conducive for the Ct10201isolate,12L:12D condition was the optimal condition for its growth. Nutritional gradient screening studies demonstrated that fructose could significantly enhance the growth rate of Ct10201as well as maltose. However, Xylose was an obvious inhibitor for its growth. Ct10201isolate cultured in alanine, leucine and nitrogen-free conditions grew well. In addition, Ct10201isolate has no special requirement on vitamin.Secondly, I interestingly found that Ct10201isolate cultured for2days after2-day-preculture in liquid medium could produce a large number of uniform short mycelium, the length of134.6±65.7μm (n=50). Lots of resting spores were formed within a short period from short length mycelium tiled into Petri dishes under25℃in the dark condition. The response surface method was used to optimize the separation conditions for resting spores, the optimal conditions were the emulsifier concentration of1.68%, ultrasonic power20W, ultrasonic time45.80s. The maximum yield of resting spore reaches72.40×104spore/ml. For the further purification of the resting spores, membrane filtration method was evaluated as the best method compared with sucrose density gradient and saturated zinc sulfate floating methods. In addition, we also observed germination of resting spores isolated from membrane filtration method. The germination rate was approx.80%.Finally, to explore the possible dormancy mechanism of resting spore, we compared the intracellular soluble protein of resting spore with that of conidiospore, as well as enzyme activity of SOD, POD, CAT and PPO. The soluble protein content and SOD activities have a significant difference between two spore types. Soluble protein content of conidiospore was23.63±0.67×10-8mg per spore, ten folds than that of resting spore. Likewise, the total activities of SOD in conidiospore was greater than that in the resting spore. Meanwhile, POD, CAT and PPO contents in either conidiospore or resting spores, have not been detected totally. Furthermore, results from using SDS-PAGE for soluble protein showed that conidiospore and resting spore have many respective specific protein bands. Analyzed by software Quantity One■, there were five specific bands in SDS-PAGE of conidiospore soluble protein, which molecular weight were41KDa,36KDa,29KDa,19KDa,15KDa, respectively. Simultaneously, there were three specific bands in SDS-PAGE of resting spores soluble protein, which molecular weight were56KDa,32KDa,16KDa, respectively. Different protein bands illustrated by SDS-PAGE from conidiosproe and resting spore meant the variant gene expression diversity in two spore types.