Study On Biological Characteristics And Domestication Optimized Cultivation Of Cordyceps Tenuipes

Cordyceps is a kind of fungal resources with important edible,medicinal and health functions.They have important application values in the fields of medicine,food industry,agriculture and modern biotechnology.Due to the scarcity of wild Cordyceps resources and the large market demand,coupled with the acceleration of urbanization and industrialization in recent years,the degradation and destruction of the natural environment in which Cordyceps grows,Cordyceps resources are indiscriminately harvested,and the output of wild Cordyceps has been declining year by year,far from being satisfied market demand.Therefore,there is an urgent need for artificial domestication and cultivation of Cordyceps,research and exploration of its related cultivation and production techniques,to improve the yield and quality of Cordyceps,or to develop new resources or new varieties of Cordyceps that can be cultivated artificially.At present,in addition to the common cultivated Cordyceps militaris,Isaria cicadae,Tolypocladium guangdongense and a few species of Cordyceps,Cordyceps tenuipes is a rare wild Cordyceps species with high economic value.There are also some researches on artificial cultivation,but due to the low yield and other reasons,there are no commercial products of its cultivation on the market.Cordyceps tenuipes is rich in a variety of effective active ingredients and has a good therapeutic effect on various diseases in the human body.It is an excellent substitute for Ophiocordyceps sinensis.At present,the research on the biological characteristics of Cordyceps tenuipes is not comprehensive,the artificial cultivation technology is not yet mature,and the yield and biological efficiency are low,which restricts its further development.This research conducted a series of studies on a wild Cordyceps collected from Huangshan Mountain.First,the morphological characteristics and molecular biology were identified,and the morphological characteristics and molecular sequence were combined to determine that it was Cordyceps tenuipes.Secondly,the biological characteristics of the mycelium of Cordyceps tenuipes were comprehensively studied.Orthogonal experiments screened the growth factors such as the optimum growth temperature,p H,carbon source,nitrogen source,etc.in the mycelial solid plates and liquid shake flasks.At the same time,preliminary explorations were made on the growth cycle of Cordyceps tenuipes,and different culture materials were screened.The effects of mycelium,primordium and spore bundle growth and yield.Further study the effects of adding silkworm pupa powder and the amount of addition on the different growth stages and yield of Cordyceps tenuipes,which has certain reference value for large-scale cultivation or commercial application.The main detailed research contents are as follows:1.Morphology and molecular identification of wild Cordyceps.In addition to the morphological characteristics of Cordyceps tenuipes,the tested ITS sequence of this Cordyceps has a sequence similarity of 99.64%with the sequence number MH857959of Cordyceps in Gen Bank.At the same time,a phylogenetic tree was constructed,and the genetic distance between it and other Cordyceps was analyzed from the perspective of genetic evolution,and it was identified as a wild Cordyceps tenuipes.2.Study on the biological characteristics of Cordyceps tenuipes mycelium.The growth curve of the mycelium of Cordyceps tenuipes was studied using PDA and PDB medium.The growth rate of the hyphae in PDA medium was not much different at different times,and the hyphae grew over the whole medium（90mm）at about 27 days.The mycelium in PDB medium is the best on the 6th day.The effect is best when the inoculation amount of the liquid shake flask is 10%.Through the single factor experiment,the optimal temperature for the growth of Cordyceps tenuipes mycelium under the solid and liquid conditions has been preliminarily screened out,PH,carbon source and nitrogen source.It is found that the optimal growth factors of Cordyceps tenuipes mycelium under solid and liquid conditions are the same,that is the optimal temperature is 25℃,the optimal p H is 7,the optimal carbon source is sucrose and the nitrogen source is yeast powder.The optimal growth factor and medium formula selected by the three-level three-factor orthogonal test were accurately verified,and the optimal medium formula was found to be A₂B₃C₃,namely sucrose 30g/L,yeast powder15g/L,KH₂PO₄2g/L and Mg SO₄·7H₂O 2g/L,the mycelial growth rate was 0.223cm/d,and the dry weight of mycelial bulb was 27.104 g/L,which were higher than the data of each experimental group.3.Research on the cultivation substrate and growth cycle of Cordyceps tenuipes.The research of cultivation substrate and growth cycle is divided into mycelium stage,primordium stage and coremia stage.The mycelium stage takes about 7 days,the color can be completed by about 3 days of light,the primordium induction requires 4 days from inoculation.From inoculation to harvest,the entire growth cycle of Cordyceps tenuipes is about 40 days.Preliminary screening of the artificial cultivation medium for Cordyceps tenuipes,rice and wheat are more suitable cultivation media,while corncobs are not suitable for the cultivation of Cordyceps tenuipes.By screening the effects of different cultivation materials on the growth of mycelium,primordium and coremia of Cordyceps tenuipes,the yield of coremia of wheat culture material with silkworm pupa powder was the best,with an average yield of 13.564g per bottle,and an average biological efficiency（67.820±6.018）%.The effect of the addition of silkworm pupa powder in the wheat culture medium on the growth of Cordyceps tenuipes was further studied,the overall growth status of coremia was better and the yield was higher when the addition amount of silkworm pupa powder was 1.5～3.0g/bottle.When the additive amount is 3.0g/bottle,the output reaches the maximum value of 18.523g,and the biological efficiency is（92.613±6.524）%.At the same time,it is found that,excessive addition of silkworm pupa powder（greater than 4.5g/bottle）will cause the mycelium to not penetrate the culture medium,which will easily cause pollution in the later stage and reduce the quality and yield of Cordyceps tenuipes.