The Research On The Characteristics And Subculture Of Armillaria Spp.symbiosis With Gastrodia Elata

Armillaria（Fr.:Fr.）Staude spp.were a type of macro fungus with extremely high medicinal and edible value.Several species of Armillaria have been identified as symbionts of the traditional Chinese medicine Gastrodia elata BI.and Polyporus umbellatus as the only nutrient source during the asexual reproduction stage of G.elata,the species and growth status of Armillaria spp.affects the quality and production of G.elata.With the gradual maturity of G.elata cultivation technology,the artificial cultivation of G.elata has gradually scaled up,and the demand for Armillaria spp.has increased accordingly.In particular,high-quality indicators can improve the stability of strain cultivation,increase production efficiency,and ensure high-quality and high-quality production of G.elata.Objective:The M1 and M2 strains isolated from the G.elata base in Xiaocaoba Town,Yiliang County,Zhaotong City,Yunnan Province,and the F2 and F3 strains isolated from the G.elata base in Choushui Township,Fusong County,Baishan City,Jilin Province are Armillaria spp.associated with G.elata,and are widely used in the production area,but their species and optimal cultivation conditions are unknown.This paper initially identified four strains of Armillaria spp.,and through the selection of high-quality strains,optimization of culture conditions and subculture,etc.,with a view to lay the foundation for the Strain cultivation of M1、M2、F2、F3 strains associated with G.elata in the two places.The main research contents are as follows:Methods:1.By extracting DNA of M1,M2,F2,F3 strains,PCR amplification and cloning,to obtain internal transcribed spacer（ITS）,Beta-tubulin（β-tubulin）and Translation elongation factor-1alpha（Tef1-α）.Through the peak map analysis and multi-sequence comparison,the differences of the four strains sequences were analyzed.The three sets of gene fragment sequences were combined to build a phylogenetic tree for classification and identification.2.The main morphological characteristics of M1,M2,F2 and F3 strains were observed during the cultivation.The extracellular laccase activity of M1,M2,F2 and F3 strains was determined by ABTS method;the cellulase,amylase,xylanase and pectinase activities were determined by DNS method;the intracellular polysaccharide content of the strains was determined by the phenol-sulfuric acid method.3.With biomass as an indicator,the single factor method was adopted by selecting the cultivation temperature,medium p H,carbon source,nitrogen source,inorganic elements and vitamins of M1 and F3 strains;a four factors and three levels orthogonal test was designed according to the single factor select results to optimize the culture condition of M1 and F3strains.4.After the M1 and F3 strains were cultured with 6 kinds of medium in the dark for 25days,they were moved to a temperature of 15°C～20°C,a humidity of 80%～90%,scatter light,and induced fruit bodies.5.Polyacrylamide gel electrophoresis（PAGE）was used to analyze esterase isozymes with the different generation subculture strains from M1-1 to M1-10.6.The soluble protein content of M1-1 to M1-10 strains was determined by Coomassie brilliant blue method.7.The ABTS and DNS method were used to determine the extracellular laccase,cellulase,amylase,xylanase,pectinase activity of the differentsubculture strains;the phenol-sulfuric acid method was used to determine the polysaccharides contents of the strain.Results:1.The sequences of ITS,β-tubulin and Tef1-αgene fragments of M1,M2,F2 and F3strains were obtained.Multi-sequence comparison analysis showed that the differences of the ITS sequences of the four Armillaria spp.strains were small,and the differences of the Tef1-αsequences were the largest.Phylogenetic analysis showed that the M1 and M2 strains were clustered with the reference sequence of Armillaria gallica Marxm.&Romagn.HKAS85517,and the F2 and F3 strains were on the same branch as the reference sequence of Armillaria cepistipes Velen.HKAS8584.2.The change trend of the enzyme activities including the extracellular laccase,cellulase,xylanase,amylase and pectinase of M1,M2,F2 and F3 strains presented a"unimodal"form,and the enzyme activity was the highest on the 12^th day.The extracellular enzyme activities of the strains were different,especially the highest enzyme activity.The polysaccharides content of the four strains showed a low,high and low trend.The F2 and F3 strains had the highest polysaccharide content on the 8th day,followed by 1.869%and 1.599%,and the M1 and M2strains had the highest on the 12th day,2.14%and 2.27%,respectively.3.The results of single factor experiment showed that the M1 and F3 strains are suitable for growing with weakly acidic,that is,the optimal p H value 5.0～6.0,and the optimal culture temperature is 25℃.The strains grew well in the medium with monosaccharide,mannitol and soluble starch as the carbon source,but poorly with maltose and sucrose.The M1 and F3 strains grew well in organic nitrogen source yeast powder and peptone,but did not grow well in a single inorganic nitrogen source medium.Mg SO₄·7H₂O and Ca Cl₂ had no significant effect on the biomass of M1 and F3 strains.The biomass of M1 and F3 strains in KH₂PO₄ and Mn SO₄·H₂O medium was significantly lower than that of the control.Vitamins have no significant effect on the biomass of M1 strain,and have a significant promoting effect on the biomass of F3 strain.VB₁ has the most obvious effect on the biomass of strain F3.4.The main factors affecting the biomass of the M1 and F3 strains were yeast powder and glucose,and the effects of Ca Cl₂ and VB₁ on the biomass of the M1 and F3 strains were not significant.5.The No.6 medium successfully induced the fruiting body of M1 strain,and no fruiting body of F3 strain was found.Medium 1-5 did not grow fruiting bodies of M1 and F3 strains.6.The results of esterase isozyme electrophoresis showed that the esterase isozymes enzyme bands number of M1-1 to M1-10 strains were all 4,the first enzyme band was wider and the color was darker.At the beginning of the enzyme band,the width and color of the enzyme band change,but there is no significant difference in Rf value.7.The soluble protein content of M1-1 to M1-10 strains showed a downward trend with the increasing subculturing,and the soluble protein content of strains M1-8 to M1-10 was lower than that of M1-1 to M1-6.The soluble protein content of each generation of M1 strain was between 18.680 mg/m L～19.852 mg/m L,there was no significant difference（P>0.05）.8.The change trend of extracellular enzyme activity increased at the beginning and then decreased,and reached the highest value on the 12th day.The polysaccharide content showed a low-high-low trend,and the highest was on the 12th day.No significant differences were observed in statistics among extracellular enzyme activities and polysaccharides content of M1generations.Conclusion:1.The Tef1-αsequence can distinguish Armillaria gallica Marxm.&Romagn.and Armillaria cepistipes Velen.,which is difficult to identify by morphological characteristics.M1 and M2 strains were identified as A.gallica,F2 and F3 strains as A.cepistipes with phylogenetic tree analysis of sequence construction.2.The M1 strain showed the highest laccase and amylase activity,and the M2 strain showed the highest cellulase,xylanase and pectinase activity.The polysaccharide content of M1 and M2 is relatively higher that of F2 and F3.It was suggested that the polysaccharide content of Armillaria spp.had interspecies difference.M1 and M2 strains were selected as high-quality strains by comprehensive analysis.3.M1 and F3 strains grew well in medium with monosaccharide and mannitol,and the disaccharide contrarily.The main factor affecting the biomass of the strain is the nitrogen source.Organic nitrogen sources can significantly increase the biomass of the M1 and F3 strains.The optimal nutritional conditions are glucose 25 g/L,yeast powder 4 g/L,Ca Cl₂ 2 g/L,VB₁ 0.05g/L.4.The No.6 medium cultivates the fruiting body of the M1 strain,and the fruiting body induction conditions are suitable for the M1 strain.5.The esterase isozyme,soluble protein content,extracellular enzyme activity and strain polysaccharide content of the M1 strain are relatively stable within 10 times of subculture,and the activity of the strain has not changed.