| Xylaria is a fungus,which belong to the class Ascomycetes,Sphaeriales,Xylariaceae,and often grows in termite nests of the large termite subfamily.However,the growth of Xylaria is inhibited in termite nests with termites living there,and it is often found in abandoned termite nests after termite migration.Because the fruiting period of Xylaria in the ground is very short,there are few reports about the species that can becultured in large scale.Xylaria is a very valuable medicinal material,which has the functions of anti-aging,calming the mind and prolonging the life.Termitomyces belongs to Basidiomycetes,Agaricales,Lyop Hyllaceae,and Termitomyces,and is symbiotic with termites.When the ant nest is destroyed,Termitomyces does not grow.Due to the weak research on its symbiosis mechanism,it is still difficult to cultivate it artificially.Termitomyces is a rare wild edible fungus and the polysaccharide in its fruit bodies has the effect of anti-oxidation,reducing blood fat and anti-tumor.In this paper,two species of Xylaria and one species of Termitomyces were isolated from ant nests which were transferred back to the laboratory and in which the ant was dead,and a pure culture system of Xylaria was established.Therefore,in this study,ant nests,two species of Xylaria,Termitomyces fruit bodies and its pure cultured mycelia were used as materials,the qualitative and quantitative analysis of the small molecule active substances in the five materials were carried out using nuclear magnetic resonance(NMR)and gas phase mass spectrometry(GC-MS),the different substances of 5 kinds of materials in pairs were analyzed using Simca-p 14.0 software.This has laid an important theoretical foundation for the large-scale artificial fermentation or culture,for the development of related drugs of Xylaria and Termitomyces.The main research contents and results of this paper are as follows:1.Two species of Xylaria were isolated,and preliminarily identified as Xylaria Escharoidea and Xylaria Angulosa by combining ITS molecular markers;one species of Termitomyceswas isolatedand preliminarily identified as Termitomyces robustus var..Yuan PY by combiningthe fruiting bodies characteristics.The two species of Xylaria have been able to be cultured on a large scale,which is an important innovation of this paper and the basis for subsequent research.2.The life cycles of the two species were observed and recorded,and it was found that the morphology and growth patterns of Xylaria Escharoidea and Xylaria Angulosawere significantly different:the growth of X.Escharoidea is like a tree,first,some rhizome hyphae were germinated along the surface of medium from the inoculant,then these hyphae twisted into thick cords and grew upward,with few branches at the growth end,after fruit bodies matured,they were rod-like or tree-like,and the outer surface turned black,but the inner and the growing end of the cords remained white;the growth of Xylaria Angulosa is like a flower filament,first,many twigs or filamentous cords were first densely grown from the inoculation to the surrounding and upward,the growth end of the cords expanded first and then branched,the basal surface of the cords of the mature fruiting bodies also became black,and the interior and growth end remained white.3.NMR technology was used for qualitative and quantitative analysis of 5 kinds of materials,in which,a total of 58 kinds of small molecule active substances wereidentified,including 26 kinds of amino acids,11 kinds of sugars,5 kinds of salts,5kinds of alcohols,3 kinds of acids and 8 kinds of other kinds.X.Escharoidea,X.Angulosa,ant nest,T.robustus var fruit bodies and its mycelia were identified as 33,19,18,31 and 26 species of substancesrespectively.4.GC-MS technology was used for qualitative and quantitative analysis of 5 kinds of materials,in which,a total of 95 kinds of small molecule active substances wereidentified,including 18 kinds of amino acidsand their derivatives,25 kinds of sugars,12 kinds of alcohols,26 kinds of acids,4 kinds of nucleoside and 10 kinds of other kinds.X.Escharoidea,X.Angulosa,ant nest,T.robustus var fruit bodies and its mycelia were identified as 40,51,27,51 and 42 species of substancesrespectively.5.The comparison results of the two techniques showed that 65 substances of X.Escharoidea were detected by the two methods,including ~1H-NMR 33 species,GC-MS40 species,and 8 species in common;63 substances of X.Angulosa were detected,including ~1H-NMR 19 species,GC-MS 51 species,and 7 species in common;44substances of ant nestwere detected,including ~1H-NMR 18 species,GC-MS 27 species,and 1 species incommon;73 substances of T.robustus varfruit bodieswere detected,including ~1H-NMR 31 species,GC-MS 51 species,and 9 species in common;60substances of T.robustus va rmyceliawere detected,including ~1H-NMR 26 species,GC-MS 42 species,and 8 species in common.In general,the two methods complement each other and can detect more substances,and GC-MS is stronger than ~1H-NMR.6.Pairwise differential substanceof 5 materials were analyzed respectively,and the results showed that for the same category,the analysis results of the two techniques tended to be consistent,for example,to the group of X.Escharoidea＿vs＿X.Angulosa,the contents of different substances were significantly increased:among the11 significantly differential substances analyzed by NMR method,10 species were significantly increased,and 15 different substances analyzed by GC-MS method were significantly increased;to the group of T.robustus varfruit bodies＿vs＿T.robustus varmycelia,the content of differential substances was also significantly increased:among the 12 significantly different substances analyzed by NMR method,9 species significantly increased,and among the 17 significantly different substances analyzed by GC-MS method,14 species significantly increased;for different category,two methods,each of two methods had its own emphasis,and pulled apart the differences between taxa,for example,to the group of X.Angulosa＿vs＿ant nest,among the 10 significantly differential substances analyzed by NMR method,7 species of 8 significantly increased sugars were characteristic to ant nests,and among the 13 significantly differential substances analyzed by GC-MS,all of them were significantly decreased,and 8 of the 13 substances were amino acids;to the group of T.robustus varfruit bodies＿vs＿ant nest,similar results were also found.The above results shows the necessity and utility of the two methods combined. |
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