Screening And Characteristics Research Of Efficient Armitlaria Mellea Strains Symbiotic With Zhaotong Gastrodia Elata

Armillaria mellea （Vahl:Fr.） P.Kumm.is a kind of higher fungi andwell-known edible fungus with important economic value and high medicinal value,excepting some strains causing ceitocybe bescens of trees.It is also an essential fungalsymbiont for the cultivation of Gastrodia elata and Grifola umbellala,andthe quality of A.mellea affects their products directly.Yunnan zhaotong is recognized as authenticproducer of high-quality of Gastrodia elata because of its unique conditions in geographyand resources in vegetation.This Study focused on screening efficient A.mellea from15strains isolated from Zhaotong,optimizing cultivation condition,affinity research with G.elata and so on,which would lay a foundation for the foster of A.mellea and industry ofG.elata in Zhaotong to modernization,professionalizition and scale.Results of thisexperiment are as follows:（1） Comparing the growth characteristics of15Armillaria spp.symbiotic with G.elata isolated from Zhaotong to1strain from other place,the results showed that therewere significant differences in the growth features among these strains.Orders from highto low were jing-234,SNA04,SNA01,SNA11,SNA03,SNA09,SNA05,SNA02,SNA14,SNA10,SNA15,SNA12,SNA13,SNA07,SNA06;The strainSNA04performed best and followed by SNA01,SNA05,SNA09,SNA11and SNA02regarding to the germination time of mycelial and rhizomorph,rhizomorph color,rhizomorph branching condition,fluorescence intensity and other growth characteristics.Accordingly,the characteristics of SNA04was the best and SNA03was better comparingto the15Armillaria spp isolated from Zhaotong.（2）Selecting2germination strains,2kind of fungal logs and8strains of A.mellea tocultivate with G.elata, then to determine the germination of G.elata seeds, output andquantity of G.elata. The experiment results showed that seed germination rate of G.elatawas better after sowing with SNm-01;output and quantity of G.elata were also betterwhen the fungal log was fungal logⅡand the A.mellea was SNA04.The“Mycenadendrobii-SNA09-fungal logⅡ”combination had the highest yield ofG.elata（1.63Kg/hole）, The“Mycena dendrobii-SNA04-fungal logⅡ”combination hadthe highest numbers of G.elata（205/hole）. （3） The comparison result of ITS sequence analysis of SNA01,SNA04and jing-234was shown that the homology of ITS sequence between SNA01and Armillaria sinapinawas96%,the homology of ITS sequence between SNA04and Armillaria gallica was99%,the homology of ITS sequence between jing-234and Armillaria mellea was97%.SNA01as Armillaria sinapina,SNA04as Armillaria gallica,jing-234as Armillariamellea were preliminary identified.（4） The best growth conditions of2Armillaria mallea strains （SNA03,SNA04） weredetermined by single-screening and orthogonal test and the biomass was regarded as theinvestigated index.25℃was the optional temperature and rhizomorph grew most quicklyunder dark cultivation;monosaccharide （glucose） and organic nitrogen （yeast extracts,protein peptone） were the optional carbon source and nitrogen sourcesseparately,inorganic salt （potassium for SNA03,potassium phosphate for SNA04） weremore favorable for rhizomorph growth than others and VB2was the best growthfactor.A1B3C1D1（Glucose︰yeast extract︰K₂SO₄（SNA03）/KH₂PO₄（SNA04）︰VB2=15g/L︰3g/L︰2g/L︰0.005g/L） was the best combination of nutritional conditionfor2stains growth,nitrogen source （yeast extract） was the most important factor in thegrowth of rhizomorph.（5） On the basis of the results of the previous Plackett-Burman experiment,responsesurface methodology was used to analyze and optimize the culture condition forRhizomorph of Armillaria mellea.Factors of the best culture medium were glucose46g/L,ethanol24g/L,yeast extracts5g/L,protein peptone13g/L,K₂SO₄2g/L,KH₂PO₄1g/L,VB20.03g/L and VB10.01g/L.Thrice repeated model yield verification were4.86g（Y₁）,4.62g（Y₂） and4.89g（Y₃） respectively,average value （4.79±0.15g） was close tothe predicted value4.88g.The experiment verified and confirmed the stability of themodel.（6） Using SNA04as the A.mellea,oak branches as the main culture medium,glucose,ethanol,yeast extracts,protein peptone and potato juice as the supplementarynutrinent sources to cultivate fungal sticks of A. mellea in bottle,results showed that strainsgrew with multiple branches,stout,white complexion and grew faster than initial one afterdroping into the supplementary nutrient source.Especially,the growth speed of rhizomorph was fastest when adding potato juice （250g/L）,which reached to1.29cm/d,faster by0.48cm/d than control group.Potatoes were the main producs withhigher yield in Zhaotong,which could be used as supplementary nutrient source tocultivate A.mellea.（7） Because A.mellea is easy to degrade,it’s essential to take different methods topreserve them.we found that the survival rate of strains could reach100%over2yearspreserving under4℃with methodsⅢ.Vitality of those serious deteriorated strains wereimproved in certain degree after rejuvenation by different media.The strains with whitecomplexion, stout and grew faster1.98times compared with control group on NO.1medium.