Effects Of Endophytic Fungus KLBMPSYLp8 On Growth And Quality Of Dendrobium Officinale And Its Molecular Mechanism

Dendrobium candidum(Dendrobium officinale Kimura et Migo),belonging to Dendrobium of Orchidaceae,is an endemic rare and endangered medicinal plant in China.It contains polysaccharides,dendrobine,flavonoids and other active medicinal ingredients,and has been widely used in health care and the treatment and prevention of a variety of diseases.It is a valuable medicine and food homologous plant.However,due to the increasing market demand,the yield and quality of Dendrobium candidum have become a hot spot of people’s attention.In the past decade,people began to focus on the study of endophytes.It was found that endophytes can not only affect the growth and metabolism of host plants,but also promote the growth and development of non-host plants and the accumulation of active ingredients.In this paper,the target strain is the endophytic fungus KLBMPSYLp8 isolated from the fruit of the tea tree(Camellia sinensis(L.)O.Ktze)and identified as the Wickerhamomyces spp.Firstly,the potential growth promoting activity of KLBMPSYLp8 and its effect on model plant Arabidopsis thaliana were explored,and then its effect on the growth and metabolite accumulation of Dendrobium candidum was studied.Based on the transcriptome data,the gene expression changes of Dendrobium candidum induced by KLBMPSYLp8 were analyzed,and the molecular mechanism of KLBMPSYLp8 promoting the growth and metabolite accumulation of Dendrobium candidum was preliminarily discussed.1、To explore the potential life promoting activity of KLBMPSYLp8 by detecting the ability of phosphate solubilization and amino acid production.KLBMPSYLp8 had the best phosphate solubilizing capacity at 38 h after inoculation,with the phosphate solubilizing capacity of 76.12 mg/L.On the 8th day after inoculation,the ability to produce tryptophan was the strongest,with the tryptophan content of 16.7mg/L.What’s more,asparagine,alanine,histidine,arginine,α-aminobutyric acid and other amino acids that are closely related to plant seed germination and root development were detected in the bacterial suspension of KLBMPSYLp8,indicating that KLBMPSYLp8 has potential promoting activity.2、The growth indexes and some physiological indexes of Arabidopsis thaliana symbiotic with KLBMPSYLp8 were determined,and the growth related genes were verified by q RT-QCR,the results showed that KLBMPSYLp8 could promote the growth of Arabidopsis thaliana to a certain extent.In co-cultivation plate of Arabidopsis and KLBMPSYLp8,the fresh weight and the number of lateral roots of Arabidopsis increased by 150.0% and 276.9% respectively compared with the control group.Similarly,when the plants were grown together with KLBMPSYLp8 in separate culture dish,the fresh weight and the number of roots increased by 165.5% and 312.5% respectively compared with the control group.In the experiment of exploring the active substances of KLBMPSYLp8 fermentation broth,the fresh weight of Arabidopsis thaliana grown in ethyl acetate extract and n-butanol extract increased by 29.7% and 24.3%,and 3-5 lateral roots grown.In the pot experiment,the fresh weight,dry weight and stem length of Arabidopsis thaliana increased by 54.5% to 355.6%,117.4% to 242.9% and 90.0%respectively from 7 to 35 days after transplanting.In addition,the chlorophyll content and soluble sμgar content also increased by 9.7% to 22.7% and 6.3% to 39.0% respectively.Some genes related to lateral root formation and auxin synthesis and transportation for q RT-QCR verification,it also proved that KLBMPSYLp8 can promote the growth and development of Arabidopsis.3、After KLBMPSYLp8 was inoculated into the aseptic seedlings of Dendrobium officinale for a period of time,it was found that the fresh weight,dry weight and the accumulation of metabolites of Dendrobium candidum were increased.And the longer the inoculation time is,the more obvious the effect get.At 9 weeks after inoculation,the fresh weight and dry weight of Dendrobium candidum in the experimental group increased by 16.1% and 47.3% respectively.In terms of metabolites,the polysaccharide content,dendrobine content,flavonoid content and polyphenol content of the experimental group of Dendrobium candidum increased by 168.3%,157.8%,298.4% and122.3% respectively compared with the control group.4、Transcriptome sequencing technology was used to detect the changes in the transcriptome of Dendrobium candidum with KLBMPSYLp8 treatment.A total of Clean Data 101.45 GB is obtained.The Q30 distribution of each sample ranged from 94% to95%,and the GC content was about 46%.The differential expressed genes of strain KLBMPSYLp8 inoculated for 0.5h,6h,24 h and 14 d were analyzed,and the number of them was 1583,1543,624 and 2223,respectively.The plants treated with the strain for14 days were selected for GO enrichment and KEGG pathway enrichment analysis.The differentially expressed genes were mainly enriched in glucose metabolism pathway,amino acid metabolism pathway and phenylpropane biosynthesis pathway.In addition,in terms of transcription factor analysis,AP2/ERF-ERF,NAC,b HLH and other transcription factors that play an important role in the biosynthesis of secondary metabolites and stress resistance are the main transcription factors annotated by differential genes.6 genes related to Dendrobium growth and 7 genes related to metabolites were selected for q RT-PCR verification,and the results were consistent with those of transcriptome analysis.In this study,the potential promoting growth activities of KLBMPSYLp8 was studied,and the effects of KLBMPSYLp8 on the growth and metabolite accumulation of tissue culture seedlings of non host plant Dendrobium candidum were analyzed.The molecular mechanism of KLBMPSYLp8 was preliminarily discussed,which laid a theoretical foundation for solving the problem of resource shortage of drug and food homologous plant Dendrobium candidum.