| Armillaria mellea,a symbiotic fungus of Gastrodia elata,has a high ecological and economic value.The growth and development of Gastrodia elata must be fully dependent on Armillaria mellea to provide necessary nutrients for its growth.Due to the increase in the use time of Armillaria mellea and the improper cultivation management of Gastrodia elata growers and producers,serious functional degradation and other adverse phenomena occurred in the recycling use of fungus Armillaria mellea.Finding and obtaining advantageous Armillaria mellea has become one of the goals of China's Gastrodia elata industry.This experiment mainly through the following several aspects of scientific research,screening and development of excellent armillaria strains for Gastrodia elata production.Firstly,through molecular identification of seven armillaria,the scientific classification status and genetic relationship among the seven armillaria were determined.Second,in order to solve the above problems,this study screened the growth conditions of the seven Armillaria mellea strains,explored the growth characteristics of the seven Armillaria mellea strains,and screened out the excellent conditions suitable for the growth of Armillaria mellea strains.Thirdly,seven strains of Armillaria mellea and three types of Gastrodia elata were mixed and planted,that is,adaptability study with Gastrodia elata,and strains with high adaptability with different types of Gastrodia elata were screened out.Based on the above experimental results,some theoretical basis and technical support were provided for the screening of high quality Armillaria mellea strains from cultivation of Gastrodia elata.The main research results of this experiment are as follows:1.Molecular identification of seven strains of Armillaria mellea.The DNA of seven strains of Armillaria were extracted through experiments,and the sequences of R DNA-ITS and R DNA-TEF were amplified to construct phylogenetic trees,and the species identification and phylogenetic classification of seven strains of Armillaria were analyzed.Strains 1,2,3,4 and 5 gathered together with Armillaria aotearoa(NZFS 242),Armillaria altimontana(Mac4),Armillaria calvescens(ST18),Armillaria gallica(M70)and Armillaria nabsnona(HKAS85523)to form a family.There was a close phylogenetic relationship among Strains 1,2,4 and 5,while strain 3 only had ITS gene,so it was necessary to compare the differences in basic group and morphological characteristics between them to determine their species.Strains 6 and 7 were clustered with Armillaria mellea(Aftol-ID 449)and Armillaria mellea(MEX88),in which strains 6 formed a well-branched(70%)sister line with Armillaria mellea(Aftol-ID 449).2.The single factor test was used to screen the growth conditions of seven Armillaria mellea strains with the biomass of the cord as the main index of the comprehensive investigation.The results showed that Armillaria mellea had the best comprehensive utilization rate of glucose,the medium with glucose as carbon source could effectively promote the growth of Armillaria mellea,and the average dry weight of the rhizomorph was the highest overall,while all strains had the worst comprehensive utilization rate of sucrose.The medium with peptone,yeast extract and beef extract as nitrogen source was beneficial to the growth of mycorrhidia,of which peptone was the best,while the medium with sodium nitrate inhibited the growth of Armillaria.Potato culture medium is most suitable for the production of Armillaria mellea.The optimum temperature range for the growth of all the seven strains was between 15? and 25?.In the dark culture environment,the rhizomorph grew well;in the acidic condition,the rhizomorph grew well and were sturdy;in the alkaline condition,the growth of the rhizomorph was inhibited and the strains were prone to death.3.The yield trend of the same strain was basically consistent with that of the three types of Gastrodia elata mixed with cultivation methods of sexual reproduction or asexual reproduction.The yield of G.elata Bl.f.elata > G.elata Bl.f.viridis Makino > G.elata Bl.f.glauca S.Chow,and the yield and quantity of the no.1 and no.5 strains were stable and had better effects after mixed with the three types of Gastrodia elata.The results showed that no.6 and No.7 strains were prone to "empty nest" when mixed with Gastrodia elata,indicating that no.6 and No.7 strains were not suitable for the cultivation and production of Gastrodia elata.The yield of Gastrodia elata cultivated by asexual propagation was significantly higher than that by sexual propagation.4.Through the determination of the effective ingredients of Gastrodia elata,the results show that whether it is sexual reproduction or vegetative reproduction,the moisture content and ash content of all strains and three types of Gastrodia after mixed planting have reached the requirements of the Chinese Pharmacopoeia(2015 edition).The same strain with the same type of Gastrodia elata mixing plant,Gastrodia elata effective components of asexual cultivation way cultivated Gastrodia elata extract content and polysaccharide content general than sexual reproduction mode cultivated Gastrodia elata,and rhizoma gastrodiae asexual cultivation mode,all the strains with three types of Gastrodia elata plant extract content have reached the China pharmacopoeia(2015 edition),and sexual reproduction cultivation mode some strains mixing plant Gastrodia elata extract content has not reached the China pharmacopoeia(2015 edition).5.Under the comparison of two kinds of cultivation mode,the use of cultivation of Gastrodia elata asexual cultivation mode,can not only improve and guarantee the output of Gastrodia elata,quality and far more than the China pharmacopoeia(2015 edition)of the provisions of the content of Gastrodia elata standard,whether on the yield or quality,asexual cultivation ways are better than that of sexual reproduction way of cultivation.Under the mode of sexual reproduction,the total contents of gastrotin and p-hydroxybenzyl alcohol were higher than those of the control strains when the seven strains were mixed with G.elata Bl.f.elata and G.elata Bl.f.glauca S.Chow,while the total contents of Gastrodia elata and p-hydroxybenzyl alcohol were the highest when the control strains were mixed with G.elata Bl.f.viridis Makino.Under the clonal propagation and cultivation method,the total amount of gastrotin and p-hydroxybenzyl alcohol of Armillaria No.1 to No.7 mixed with G.elata Bl.f.elata was higher than that of the control strain.The total amount of gastrodin and p-hydroxybenzyl alcohol in the mixed planting of Strains No.1,No.2,No.3 and No.5 are higher than or equal to the control strain.The total amount of gastrodin and p-hydroxybenzyl alcohol in No.1 and No.5 strains mixed with G.elata Bl.f.viridis Makino was significantly higher than that of the control strains.Therefore,strains 1 and 5 not only showed stable and good performance in terms of yield and quantity,but also had high quality of medicinal materials,and the total amount of gastrodin and p-hydroxybenzyl alcohol could far exceed the provisions of The Chinese Pharmacopoeia(2015 edition). |
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