Growth Conditions And Diversity Of Fungus Mycena Spp. For Gastrodia Elata Germination

As a kind of homologous plant food and medicine,Gastrodia elata Bl.not only has the medicinal value of improving the function of heart and brain,antiaging,and anticancer,but also has the nutritional value of supplemental trace elements and amino acids.As an important Chinese medicinal material,G.elata has a large demand in China and abroad,thus lead to a shortage of demand in the market.G.elata is a highly degraded orchid,and it can not germinate without the help of the germination of fungus.Furthermore,it has no root to absorb nutrients from the external environment in the later period of life cycle and needs to grow well with the symbiont of Armillaria mellea.Therefore,the cooperation of germination fungus and Armillaria mellea to promote seed germination and vegetative growth play a decisive role in the growth process of G.elata Bl.At present,there are some problems,such as strain degeneration and pollution,greatly limit the G.elata Bl cultivation and the development of its industry.Moreover,for the focus of this article,the fungus(Mycena)for the germination of G.elata Bl,there are still some problems,such as confusion of species identity,difficulty of isolation and identification,as well as the delayed culturing period of the associated fungi.To solve these problems,four studies are carried out in this subject: high-throughput sequencing,specific primers design for Mycena,the optimization of the medium and fungal isolation.The following conclusions are obtained:1.Through high-throughput sequencing,it was found that there were some differences in community structure among three stages of the early germination of G.elata Bl and its living soil.Among the four groups,there were 239 different OTUs,of which 16 were common in the four groups,and the community structure of the protocorm was more complex.There are 8 genera only distributed in the protocorm,7 genera both distributed in the protocorm and the phase II of GE growth,but only 1 genus distributed in the three stages.Mycena rarely distribute in the test samples,the proportion of distribution in each group decrease with the growth of protocorm,the proportion in the protocorm samples account for 1.5%,the proportion in the phase II of GE growth samples account for 0.05%,and it is not detected in the phase ? of GE growth samples.The proportion in the soil samples is extremely trace.So the material using for Mycena isolation needs to be greatly screened and needs a lot.These conclusions laid the foundation for further targeted isolation materials.2.Five pairs of the specific primers for Mycena were designed,and their specificity,sensitivity and practical application potential were evaluated.Among them,four pairs of primers with good specificity were M52-2,M22-3,M11-4 and BZ-5.The amplified proportion of 125 test strains from 3 classes and 45 families were 2.4%,6.4%,4% and 7.2%,respectively.The sensitivity of M22-3 and BZ-5 was good and could detect the minimum concentration of mycelium in the soil for 0.033%.These two primers can also successfully detect Mycena from the total DNA samples obtained from soils and other biological materials,in which Mycena may exist.The design of specific primers provides a fast and simple method for identification of Mycena and can improve the problem of identification and confusion of Mycena strains.3.The synthetic medium and natural medium for the culture of first class seed of Mycena were designed by uniform design,and the synthetic medium was optimized by quadratic polynomial stepwise regression,the medium for the rapid growth of Mycena was obtained.After 21 days of cultivation of Mycena with the synthetic medium,the colony diameter of the optimum formula was 69.0cm,and the biomass of biomass optimization formula was 0.2685 g.After 15 days of cultivation of Mycena with the natural medium designed by uniform design,the colony diameter of maximum diameter formula was 74.7cm,and the biomass of maximum biomass formula was 0.3653 g.The interaction between several nutrient factors which had great influence on the growth of Mycena was discussed.The main factors affecting the diameter of Mycena colony were glycine,potassium dihydrogen phosphate,soluble starch,yeast extract and peptone,among which,Potassium dihydrogen phosphate has strong interaction with soluble starch,and yeast extract has strong interaction with peptone.The factors affecting the biomass of Mycena colony are mannitol,epsom salt,sucrose,glycine and peptone,among which,sucrose had strong interaction with mannitol and glycine respectively,and epsom salt had strong interaction with peptone and mannitol respectively.The optimization of culture medium provided a theoretical basis for studying the early inoculation medium of Mycena.4.In the end,259 fungal strains were isolated from a variety of samples.After morphologically being categorized,65 OTUs were sequenced,and these strains belonged to 3 classes and 39 families,of which the ratio of Ascomycota(93.2%)and Basidiomycota(6%)is max,and Mucoromycota accounted for less than 1%.Some fungi annotated in high-throughput sequencing have been also obtained in traditional fungal isolation,including Mariannaea(3%),Trichoderma(8.3%),Clonostachys(4.5%),and Fusarium(15.8%).It is presumed that these fungi may be potential germination fungus and the germination test needs to be carried out to verify in the later period.In a summary,the results are expected to shorten the growth time of Mycena,lay the foundation for the rapid positioning and isolation of Mycena,and to promote the development of G.elata Bl.industry in the future.