Studies Of Endophytic Fungus On Promoting Quality Of Dendrobium Officinale And Its Molecular Mechanism

BackgroundDendrobium officinale was a rare traditional Chinese medicinal herb and topped the list of "Nine Chinese fairy herbs",which had the advantage of relieving stomach upset,promoting the production of physical fluid,expelling pathogenic heat and nourishing Yin of body.Currently,cultivation areas of living tree and cliff attached patterns were developing rapidly and their materials quality and economic benefits were much more than common patterns.More attention has been paid to the mechanism of growth,development and ingredient metabolism of plants in the attached pattern because of the cultivation features with no fertilizers and pesticides in natural environment.As an essential bio-agent,the endophytic fungus had played important functional roles on growth,development and ingredient metabolism to medicinal herbs.Currently,the studies on the effects and their active mechanisms of endophytic fungus on medicinal herbs are still not deep enough.To a certain extent,it has been restricted that the realization of the effects of endophytic fungus on the quality of traditional Chinese medicine and the application of endophytic fungus in production and cultivation of medicinal herbs.In this study,the superior endophytic fungus from D.officinale which could promote growth and ingredient metabolism of host were screened and their symbiotic system with seedlings was established.Meanwhile,the functional role of water-soluble elicitors and their main active fraction from superior strains was studied.Variation of polysaccharides contents of seedlings after infected by endophytic fungus was discussed.Furthermore,the molecular mechanism of polysaccharides accumulation was revealed by RNA-Seq and bioinformatics studies.It is hopeful to provide experimental evidences and scientific basis for elucidating the molecular mechanisms of endophytic fungus afftecing the quality of Chinese traditional medicinal and microbial resources on quality enhancement and applied biotechnology for D.officinale.Methods(1)Fifty-two endophytic fungus strains isolated from D.officinale before were inoculated to seedlings.The strains which could maintain long-term stability of symbiotic system were preliminarily selected.Moreover,the strained were inoculated again to study the effects on yields,growth characters and active ingredient contents including polysaccharides,mannose and ethanol-soluble extractives contents of seedlings.The three ingredient contents were measured by phonel-sulfate method,pre-column derivatization HPLC(high performance liquid chromatography)and hot dipping method respectively.Then superior strains which could significantly promote growth and ingredient metabolism of seedlings were screened.(2)The mycelium of superior strains was sterilized and smashed to be the water-soluble elicitors(Extract of mycelium EM)after cultured in PDB(potato dextrose broth)medium to a certain stage.The soluble sugar contents of EM at different stages(uncultured,2nd,4th,8th,12th,16th and 20th day)were measured by anthrone colorimetry method.Based on the soluble sugar contents,low(300 mg·L-1)and high(600 mg·L-1)concentrations elicitors of superior strains were produced and added to symbiotic medium of seedlings.Yields,growth characters,polysaccharides,mannose,ethanol-soluble extractives,Naringenin contents of stem,total flavonoids,total phenols and Schaftoside contents of leaves were studied.The Naringenin and Schaftoside contents were measured by UPLC(ultra performance liquid chromatography)and HPLC means,while the total flavonoids and total phenols contents were measured by UV(ultraviolet spectrophotometry).ES,SL and PPF fraction of elicitors were separated by modern chemical separatation means and added to medium of seedlings.The functional role on growth and ingredient metabolism of seedlings among different fractions(ES、SL、PPF),PPF from different strains and different concentration from one strain(PPF30、PPF60、PPF120、PPF240 contained PPF30、60、120、240 mg·L-1)were compared respectively.(3)Variations of the polysaccharides,mannose,ethanol-soluble extractives,Naringenin contents of stem,total flavonoids,total phenols,Schaftoside contents and DPPH(1,1-diphenyl-2-picrylhydrazyl)radical scavenging capacity of ethanol-soluble extractives from leaves of seedlings infected by DO14 at different time(uninfected,3rd,6th,15th,21th and 30th)were studied.Based on the polysaccharides contents,the RNA of IFP,UFP,SCF and ICF sample at 6th were extracted and used to RNA-Seq analysis including cDNA library construction,paired end sequencing,quality test of reads and De novo assembly.The unigenes gained were compared with the public database Swiss-prot,NR,Pfam,KOG,KEGG and their CDS and ORF were predicted by GENScan software to analyze the overall expression level.Differentially expressed genes(DGE)were screened by log2FC(Fold change)and DGE referred to glycometabolism were screened with expression level equal or greater than 5.0 and GO and KEGG functional annotation were carried.The reverse transcription of IFP and UFP were taken by PrimerScript RT kit and Real-Time PCR reaction of DGE referred to glycometabolism were carried with β-actin as the reference gene,relative expressed level was calculated by 2-△△Ct method.The IC50 value was analyzed by GraphPad Prism software,the data were processed by Excel 2010 and SPSS 17.0 software with one-way ANOVA and independent sample tested.Results(1)Most seedlings of D.officinale became etiolation and bleaching or overwhelmed by endophytic fungus,only DO12(Nemania sp.),DO14(Pestalotiopsis sp.),DO18(Talaromyces sp.),DO19(Xylariaceae sp.),DO34(Phialocephala scopiformis),DO38(Tolypocladium sp.),DO120(Hypoxylon sp.)could grow symbiotically with seedlings in long time.The symbiotic system with improved banana MS medium at 10 h·d-1 of illumination time,1500 Lux of illumination intensity at 25℃ was established to further study on the functional role of 7 strains above.Lastly,four superior strains DO14,DO18,DO19 and DO120 were selected,which could significantly promote growth and active ingredient contents of seedlings.They could promote yields 5.57± 0.46%,4.40 ± 0.37%,64.90± 5.79%and 1.42 ± 0.25%,polysaccharides content 114.34± 13.2%,70.72±6.42%,196.02±18.68%and 157.37± 13.71%,mannose content 2.21 ±3.34%,105.79±7.60%,160.72±15.55%and 132.02± 11.0%,while DO18 and DO19 could promote ethanol-soluble extractives content 4.33±0.52%and 6.27±0.43%respectively.(2)The soluble sugar contents of elicitors from DO14,DO18,DO19 and DO120 were the highest when cultured for 4 days,the contents were 1.530±0.124,3.484±0.142,1.188±0.085 and 3.015±0.277 mg·mL-1 respectively.The low(300 mg·L-1)and high(600 mg·L-1)concentration elicitors from 4 superior strains could promote yields,polysaccharides and mannose contents.Some elicitors could promote ethanol-soluble extractives,total flavonoids,total phenols,Naringenin and Schaftoside contents.While the effects of DO14EM-300 to yields.DO14EM-600 to polysaccharides and Schaftoside,DO19EM-600 to mannose and Naringenin,DO19EM-300 to total flavonoids and total phenols、DO120EM-600 to ethanol-soluble extractives contents were the best showing 55.27±4.39%.86.41±7.97%,30.57±2.64%,98.92±7.87%,18.85±2.01%,16.48± 1.37%,5.99±0.49%and 8.99± 1.01%higher than control.The effects on promoting growth and ingredient metabolism of ES and PPF were better than SL while the effects of ES and PPF were equal.There was different functional role between PPF from different strains,the increasing effects of DO14PPF to polysaccharides and mannose contents was 1.90±0.11,2.90±0.18,2.15±0.19 and 2.40±0.27.6.74±0.34.4.32±0.37 times higher than DO18PPF,DO19PPF and DO120PPF respectively.Besides,there was certain specificity in the promotion effects of DO18PPF to total flavonoids,DO19PPF to total phenols and DO120PPF to ethanol-soluble extractives contents.There was different functional role between different concentrations from one strain(DO14),the effect of DO14PPF60 promoting Naringenin content was better than DO14PPF30.However,there was no significantly difference between effects to growth,polysaccharides,mannose and Schaftoside contents from different concentration of PPF.(3)The saccharides contents of seedlings started to increase significantly 6 days after infected by DO14,polysaccharides and mannose content was 17.66± 1.38%and 19.99±2.04%higher than control.Total flavonoids,total phenols and Schaftoside content was 5.42±0.53,6.68±0.28 and 0.3462±0.0240 mg·g-1 respectively which was the highest at 15 days after infected.Meanwhile,ethanol-soluble extractives and Naringenin content was 25.41±0.13%and 9.57±1.53 μg·g-1 which was the highest at 21 and 30 days after infected respectively.The DPPH radical scavenging capacity was the best at 15 days after infected while IC50 was 4.08 mg·mL-1.117869 and 58792 unigene were gained from D.officinale and endophytic fungi sample.There was 37568,62675,43999,49165,22033 unigene of D.officinale and 16235,40148,26466,13395,14275 of fungi annotated respectively when compared with Swiss-prot,NR,Pfam,KOG and KEGG database.GO function annotation showed the unigenes from D.officinale and fungi both referred to molecular function,biological process and cellar component.KEGG function annotation showed 255,252 pathways annotated and 48026,44926 CDS predicted of D.officinale and fungi respectively.There was different of expression of unigene from different biological repetition,however,ninety percent of unigene expression value distributed from 0.3 to 15.3516,7638 DGE were selected from D.officinale and fungi with 2120,3957 up-regulated and 1396,3681 down-regulated,which significantly annotated 215,172 GO items and 23,37 pathway respectively.Numbers of DGE from D.officinale and fungi referred to integral to membrane,ATP binding,extracellular region,cell wall,cellular cell wall organization and plasmodesma items.25 DGE referred to glycometabolism were screened which annotated to 4 glycometabolism pathways including starch and sucrose metabolism(ko00500),fructose and mannose metabolism(ko00501),pentose and glucuronate interconversions(ko00040)and galactose metabolism(ko00052).The data from RNA-Seq were verified accurately by Real-Time PCR.Conclusion(1)Four endophytic fungi strains DO14,DO18,DO19 and DO120 from D.officinale with significantly promoting growth and ingredient metabolism were discovered to be the superior strains for functional role study between endophytic fungi and D.officinale.(2)PPF was confirmed to be one of the main active constituents responsible for promoting growth and active ingredients accumulation in D.officinale.Besides,the optimum effects happened when treated with DO14PPF30 mg·L-1 to stem fresh weights and polysaccharides,DO14PPF60 mg·L-1 to Naringenin and Schaftoside,DO18PPF30 mg·L-1 to total flavonoids,DO19PPF30 mg·L-1 to total phenols and DO120PPF30 mg·L-1 to ethanol-soluble extractives content respectively.PPF was regarded as potential additives to promote quality and important microbial resources on effective fungus fertilizer utilization.(3)Twenty-five independent DGE referred to glycometabolism were screened from D.officinale after infected by DO14 among which twenty-four DGE were firstly reported in D.officinale.The molecular mechanism of endophytic fungus promoting polysaccharides contents accumulation was ascertained.Polysaccharides accumulation was promoted by endophytic fungus via up-regulating the key gene in starch and sucrose metabolism(ko00500)and fructose and mannose metabolism(ko00501)including the ones encoding endo-1,3-beta-D-glucosidase(EGLC),fructose-bisphosphate aldolase(ALDO)fructose-1,6-bisphosphatase(FBP),diphosphate-dependent phosphofructokinase(PFP),6-phosphofructokinase(PFK),mannose-6-phosphate isomerase(PMI)and mannose-1-phosphate guanylyltransferase(GMPP).The work provided experimental evidences and scientific basis for elucidating the molecular mechanisms of endophytic fungus affecting the quality of Chinese traditional medicinal.