| Tripterygium wilfordii is one of the precious traditional medicinal plants,which has the function of anti-bacterial,anti-inflammatory,anti-tumor,anti-HIV.However,T.wilfordii can not meet the demand of domestic and foreign markets,because of fewer wild resources and long growth period of artificial cultivation.Related studies indicated that endophytic fungi and bacteria have great impact on physiological growth of the host plants by the production of dissolving phosphorus,iron carrier and indole acetic acid(IAA),which enhances anti-reversibility of host plants and participates in synthesis the same or similar secondary metabolites of host plant.Characteristics of endophytic strains and their combinations was evaluated and the effects of endophytic strains and their combinations on growth-promoting,physiological and biochemical characteristics,synthesis of active substances were studied,through building co-culture ecosystem,and suspended elicitor adding technology.Main results are as follows.1.This study evaluated plant growth promoting traits and effect on germination of wheat seeds of two endophytic fungi,Fusarium oxysporuwm NS33 and Penicilliuz steckii NS6,two endophytic bacteria,Enterobacter cloacae sub sp LG3 and Serratia marcescens LY1,isolated from T.wilfordii,and their bacterial and fungal pair combinations NS33-LG3,NS33-LY1,NS6-LG3,NS6-LY1.Fungal strain NS6 and NS33 and strain combination NS33-LG3 secreted more IAA and siderophore.Combination NS33-LY1 showed higher levels of siderophore production and phosphate solubilization.Strain LY1 has large phosphate dissolving capability.Most of the endophytes and their combinations promoted the germination rate of wheat seeds,longation of embryo and radicle,root vigor and chlorophyll content of wheat seedlings.Relative to the single culture,the co-culture of strain NS33 and LG3 synergistically enhanced the secretion of IAA,and the root activity of wheat seedlings was also further promoted when co-inoculating this pairs.2.The target endophytic strains and their combinations were co-cultured with T.wilfordii cells,respectively.The growth and physio-biochemical properties of T.wilfordii suspension cells were studied in the various co-culture systems.The results showed that in the early stage of co-culture all the single strain promoted the T.wilfordii cell growth ant the NS6 strain exhibited the most significant effect,however,during the late period all the endophytic strains and their combinations acted a negative role on the plant cell growth.When T.wilfordii cells were co-cultured with the two endophytic fungi and the strain combinations,the pH values of supernatants were increased obviously,but for the T.wilfordii cells and LY1 strain co-culture system,the pH values were decreased.In addition,the sugar consumption of supernatants is the highest when T.wilfordii cells co-cultured with the mixed strains,and the soluble protein content was enhanced as the strains were inoculated singly.The activities of POD,CAT and SOD of T.wilfordii cells were influenced by the endophytes.Compared with the control,the single strain inoculation made T.wilfordii cells exhibit the higher activities of POD and CAT and the lower MDA content.3.The target strains and their combinations were made into bacterium liquid(JY)and bacteria(JT)elictors and then added into T.wilfordii cell suspension culture systems.All elicitors promoted the fresh weight of suspension cells and lowered pH values of culture media,particularly the elicitor LG3-JY and LY1-JY showed greater effects.Electrical conductivity of culture media was increased by the JY elicitors,however,reduced by the JT elicitor.All elicitors enhanced consumption rates of nutrient components of suspension cell culture media.The elicitors had different effects on POD,CAT and SOD activities at the different stages.The MDA content changed regularly to rise in the begining,but decrease in the end.4.Tissue culture seedlings of T.wilfordii were inoculated with the target strains and their combinations.All of these strains and their combinations improved growth of the tissue culture seedlings.The endophytic fungi had stronger positive effects than the endophytic bacteria.Compared with the single inoculation,most of mixture inoculation of the endophytic fungi and bacteria did not further enlarge or reduce the growth promoting effects on T.wilfordii tissue culture seedlings.The content of chlorophyll and soluble protein of T.wilfordii tissue culture seedlings were significantly increased by inoculating endophytes,however,the content of soluble suger reduced.Endophytes and their combinations decreased the activies of CAT,MDA,and superoxide anion free radical of tissue culture seedlings,and especially strain combinations contributed more.5.Absorption of C,N,P and K of T.wilfordii tissue culture of seedlings changed,when inoculated with the target strains and their combinations.All strains and their combinations obviously increased the content of C and K of the tissue culture seedlings.The inoculation of endophytic bacteria LG3 and LY1 promoted the accumulation of N.The single strain NS33,LG3,LY1 and the strain pair NS33-LY1 increased P content of the tissue culture seedlings.Compared with the single inoculation,mixture inoculation of endophytic bacteria and endophytic fungi had both positive and negative effects on the absorption of nutrient elements of T.wilfordii tissue culture seedlings.All endophytes and their combinations improved the accumulation of triptolide and celastrol of T,wilfordii tissue culture seedlings,and especially combination NS33-LG3 and strain LG3 showed great effects.Co-inoculation with strain NS33 and LG3,NS6 and LY1 imposed positive effects on celastrol accumulation. |
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