Study The Degenerated Mechanism Of Cordyceps Militaris Strain And The Key Cultivation Technology In Solar Greenhouse Of Xinjiang

Objective:Cordyceps militaris can be used as functional food and traditional medicine,which contains variety of beneficial bioactive components.It is an effective way to resolve the shortage of wild resources by artificial cultivation.The artificial cultivation requires the aid of agricultural facilities.The large number of solar greenhouses in Xinjiang provide plenty of resources for C.militaris cultivating.But the high frequency of strain degeneration has become the most outstanding problem during artificial cultivation.However,the degeneration mechanism is still unclear.Therefore,in this study,we focued on degeneration mechanism of C.militaris strain and exploring cultivated mode using solar greenhouse in XinJiang to provide theoretical and technical guidance for C.militaris strain selecting and cultivating.Methods:?1?Parental strains which can produce well developed fruiting bodies were screened from preserved strains.The ascospores were isolated from fruiting bodies by suspension method.The hybrid F₁strains were isolated from fruiting bodies tissue produced by cross-mating between ascospores based on mating type genes,and the strains with high yield were screened;?2?In order to improve the yield and content of cordycepin and adenosine of fruiting bodies,the F₁strains of CMS1,CMZ1 and CMX1 from top,middle and base of the same fruiting body as well as parental strains of CM1406 and CM1409 were inoculated in wheat and rice solid-substrate,respectively.Effect of light condition and low temperature stimulate on CMS1 and CMZ1 yield and contents of bioactive compounds?using HPLC?in fruiting bodies were investigated;?3?The C.militaris superior strains of CMS1 and CMZ1 were inoculated in artificial culture fruiting bodies in solar greenhouse in Xinhu 24 company of Manas county Xinjiang to explore the cultivation mode in solar greenhouse.?4?In order to study the degeneration mechanism of strain,the F1 hybrid strains were subcultured one time for every 3 months in total of 3 generations to obtain the degenerated strains.The mating type of parental strains and single asexual spores of each parental strains were detected by PCR.Then the fruiting bodies production and ratio of mating type of asexual spores of normal F₄ strains were compared.?5?To further study the degeneration mechanism,the single-asexual spore strains of CMS1-2 and CMS1-3 isolated from normal strain of CMS1?F₄?were subcultured,respectively.The morphology change of colony were observed during subculture.The single asexual spores strains were isolated from sectorization and non sectorization regions,and cross mating with single asexual spores of opposite mating type.And the 1^st,3^th and 5^th generations were sequenced by transcriptome at the primordium stage after cross mating in wheat substrates.Results:?1?Ten uncontaminated single-ascospore strains were isolated from CM1406 and CM1409fruiting bodies,seven of them were MAT 1-1 mating type,and three were MAT 1-2 mating type.Cross mating with opposite mating type of single-ascospore strains could produce abundant fruiting bodies,however,all of the single ascospore produced few or did not produce fruiting bodies.F₁ strains in total 50were isolated from top,middle and base of fruiting bodies after cross mating by combination CM1406-1and CM1409-5,respectively,84%strains were heterokaryon?containing both MAT1-1 and MAT 1-2 mating type genes?.?2?The yield of fruiting bodies of superior F₁ strains CMZ1 and CMX1 had significant difference in wheat and rice substrates,and the fruiting bodies yield were higher in wheat substrate.However the fruiting bodies of CMS1 had no significant difference on rice and wheat substrate.While the rice substrate were more favorable for the accumulation of cordycepin and adenosine in the fruiting bodies.The fruiting bodies yield and bioactive compounds contents were affected by strains isolated parts,solid-substrates,light condition and low temperature stimulation.?3?The light is necessary for fruiting bodies formation.And fruiting body yield and adenosine content were higher under red light,white light was beneficial for cordycepin accumulation.At primordium stage,low temperature of 15?for 4 h·d^-1 in total 7 days achieved higher yield and quality of fruiting bodies.After transform the solar greenhouse,the superior strains of hybrid F₁ strains were used to artificial cultivate.The red light was provided during primordium stage,then change to white light after the fruiting bodies reach to 1cm.During the fruiting bodies grown,the duration and times of aeration can be prolonged,properly.The dry weight of fruiting bodies can achieve to 38.00g and 42.94g by 400g wheat of CMS1 ang CMZ1,respectively.The bioactive compounds were higher in rice substrates.So C.militaris fruiting bodies can be artificially cultivated in solar greenhouse.?4?Among the 10 degenerated strains in F₄ strains by subculture and preservation,only one strain contains one mating type gene;the normal strain CMS1 can form fruiting bodies,the ratio of different mating type of asexual spores were close to 1:2,and the fruiting bodies production were difference between different combination of asexual spores from CMS1.But all the single asexual spores isolated from degenerated strains of CMZ1 and CMX1 were MAT 1-2 mating type.And asexual spores from CMS5 and other degenerated strains were MAT 1-1 mating type.The results showed that the mating type of different asexual spores were out of balance,severely.?5?The third generation of CMS1-3 strain?CMS1-3Z3?showed sectorization area,about 1/4 area and the colony color was faint yellow.And the single asexual spores isolated from the sectorization can produced fruiting bodies by cross mating with single-asexual spore of the CMS1-2Z3,while the single asexual spores isolated from non-sectorization area could not change color or form fruiting bodies after cross mating.The fruiting bodies yield by cross mating with CMS1-2 and CMS1-3 strain declined as continuously subcultured,and only a few undeveloped fruiting bodies formed in the fifth generation.According the KEGG functional enrichment and differential gene expression of transcriptome,we speculated that during the strains subcultured,the degeneration were mainly because genes related to sexual reproduction and fruiting bodies morphogenesis,mutations and toxic substances accumulation,as well as the reactive oxygen accumulation and the ability of elimination of activated oxygen weaked.Conclusions:Above all,cross mating by single ascospores and asexual spores between opposite mating type gene can produce abundant of fruiting bodies,which provid lots of material fro superior strains selecting.The fruiting bodies of C.militaris were formed by random combination between different mycelia,the heterocaryon were in majority.It is viable to cultivate C.militaris fruiting bodies in solar greenhouse by optimized cultivation conditions and standardize management.And in order to obtain higher yield and quality of fruiting bodies,different light at different stage and low temperature stimulation should be applied.The single-asexual spores were homokaryon,the primary reason of fruiting bodies production decline were that the ratio of different mating type asexual spores were out of balance and mutation spores produced.Sectorization is a sign of degeneration.And the mechanism of strain degeneration may the continous tissue separation the isolation of C.militaris strain,the cell viability was declined by the accumulation of harmful substances.