Enrichment Dynamics And Mechanism Of Sphingomonas In Dendrobium Officinale In Winter

Dendrobium officinale is an important traditional medicinal plant,and artificial planting is the main source at present.With the northward migration of D.officinale planting area and the development of imitation wild planting,low temperature resistance has become one of the key technologies of D.officinale planting.Based on the previous finding that D.officinale has a unique population structure in winter,the endophytic species characteristics of D.officinale in Huzhou and Shaanxi in winter and summer were compared in this paper,and then the enrichment dynamics and mechanism of dominant bacteria were studied,so as to provide a basis for cold resistance of D.officinale.Firstly,amplicon sequencing showed that the low temperature of winter significantly induced the reduction of the abundance and diversity of endophytes in D.officinale,while Sphingomonas was significantly enriched.The abundance of Sphingomonas was 62.71%and 63.55%in Huzhou,and 25.14%,13.07%in Shaanxi,respectively.However,Burkholderia-Caballeronia-Paraburkholderia was the dominant bacteria in stem and leaf in summer with the abundance of 78.14%and 75.79%in Huzhou and 73.81%and 85.09%in Shaanxi,respectively.The functional prediction based on PICRUSt2 flora showed that the functions related to amino acid metabolism and membrane transport decreased significantly in winter,while the functions related to defense increased,indicating that the growth rate of microorganisms decreased and the ability of resistance was enhanced at low temperature.Among them,Sphingomonas might play an active role in promoting cold resistance of D.officinale.Secondly,the factors inducing Sphingomonas enrichment at low temperature were investigated.Under low temperature stress,the plants of D.officinale showed significantly increased contents of H₂O₂ and O₂·^-,while the photosynthetic rate and root absorption capacity decreased significantly,which led to the increase of oxidation level and nutrient deficiency.Altogether 35 endophytes from 19 genera were isolated,including strain Sphingomonas paucimobilis ZJSH1,9 Curtobacterium strains,8 Bacillus strains and 2 Pantoea strains.Strain ZJSH1 had higher resistance to low temperature than58.82%strain,higher resistance to H₂O₂（30 m M）than 88.24%strain,and higher resistance to oligotrophic(10^-1×R2A)than 94.12%strain,which indicated comprehensively strong resistance.Cultured mixedly the 35 strains,the abundance of S.paucimobilis ZJSH1 increased with increasing levels of oxidation and oligotrophy under low temperature,while the abundance of the other strains decreased significantly or poorly.The highest abundance of S.paucimobilis ZJSH1 was 32.76%and 35.94%when cultured with 15 m M H₂O₂ and 10^-1 x R2A respectively,which was significantly higher than that of Pantoea.Thirdly,genomic analysis showed that the genome of S.paucimobilis ZJSH1contains a variety of stresses resistance genes,including two-component regulatory system,antioxidant,osmotic regulation,nutrient utilization and plant hormone synthesis.Non-targeted metabonomics analysis showed that S.paucimobilis ZJSH1 produced 5plant hormones（salicylic acid,indole-3-acetic acid,zeatin,hombrassinolide and ethephon）,4 osmotic adjustment substances（proline,spermine,spermidine and betaine）,and 5-hydroxytryptamine under low temperature stress.Comparing the metabolites at 12℃and 30℃,there were 315 differential metabolites,including 237 up-regulated metabolites and 78 down-regulated metabolites.Among them,salicylic acid（SA）,proline and spermine increased by 91.94 times,15.75 times and 6.05 times respectively at low temperature,which might promote the cold resistance of strains and plants.Finally,the effects of SA with the highest yield on the enrichment and stress resistance of strains were investigated.Under culture conditions,the growth of SA-deletion mutant（ZJSH1/△pch B）decreased significantly under low temperature and oxidative stress,while the growth of supplemental mutant（ZJSH1/△pch B-C）was close to that of wild strain（ZJSH1）,but the growth of mutant changed little under oligotrophic stress.Inoculated the wild and mutant strains with the 34 strains in the plant,the wild strain ZJSH1 was dominant in plant at low temperature with the abundance of 21.95%,31.30%and 37.31%in root,stem and leaf（LT-MZ）,respectively.The abundance of ZJSH1/△pch B in root,stem and leaf decreased to 17.58%,17.10%and 20.65%,respectively,which were significantly higher than that of ZJSH1 at normal temperature.Under low temperature stress,the relative conductivity and MDA content of plants inoculated with the wild strain were significantly lower than those inoculated with the deletion mutant,while the contents of proline and soluble sugar and the activities of antioxidant enzymes（SOD,CAT,POD）were significantly higher.It indicated that SA mediated the enrichment and cold resistance of S.paucimobilis ZJSH1 in plants.In conclusion,Sphingomonas is enriched in D.officinale in winter,which was demonstrated in mixed culture and the plant experiments with the representative strain of S.paucimobilis ZJSH1.S.paucimobilis ZJSH1 showed strong resistances against oxidation,oligotrophic and low temperature,and its metabolites such as SA play an important role in low temperature enrichment and cold resistance.These results might help develope the stress resistance theory of D.officinale and the technology to enhance the cold resistance of D.officinale.