Effects Of ACC On Rhizosphere Colonization By Pseudomonas Putida UW4

Pseudomonas putida UW4 is a typical plant growth promoting rhizobacteria(PGPR)and 1-aminocyclopropane-1-carboxylicacid deaminase(Acd S)producing strain,which can degrade ACC and decrease ethylene biosynthesis in plant.The effect of ACC on PGPR colonization in plant rhizosphere has not been understood well.In this study,P.putida UW4 and its Acd S mutant strain UW4 Acd Swere marked by GFP,the colonization traits in plant rhizosphere of the two marked strains were tested.The summary of results was as follows:1.Construction of the green fluorescent protein(GFP)expression vector pBBRGFP for triparental mating.A nonconjugative plasmid pBBR1MCS2 containing the mob gene was linearized by restriction endonuclease Eco RI and Hind III,and the 5132 bp linear vector fragment was obtained.Using green fluorescent plasmid pGFPuv as template,the target fragment of GFP gene ecpression cassette(1094 bp)controlled by lacZ promoter was cloned by PCR.Both ends of the linear vector fragment and the target fragment had 30 bases that were complementary to each other.The two fragments were ligated by using special recombinant enzyme to generate a plasmid p BBRGFP,which was able to express green protein and transfer by helper plasmid.2.Construction of the fluorescent labeled P.putida strains.E.coli DH5? with plasmid pBBRGFP was used as the donor strain,E.coli DH5? with helper plasmid pRK2013 was used as the helper strain,UW4 or UW4 Acd S-were used as the receptor strain.The fluorescent labeled strains were obtained by triparental mating,and named UW4-G and UW4 AcdS--G.The fluorescent labeled strains were determined by fluorescent testing and PCR.3.Plasmid stability and growth rate of the fluorescent labelled P.putida strains.Under the condition of non selective pressure,the green fluorescent plasmid in UW4-G and UW4 AcdS--G showed 100% stability in the continuous passage of liquid culture for 125 h.Compared with the growth curves of UW4,UW4 Acd S-,UW4-G and UW4 Acd S--G,the fluorescent labelled strains lagged behind slightly that to the original strain.4.Chemotaxis to ACC and colonization in wheat rhizosphere of the fluorescent labelled P.putida strains.All of the four strains UW4,UW4 AcdS-,UW4-G and UW4 AcdS--G showed the same chemotactic response to arginine and succinic acid.However,UW4 and UW4-G both showed strong chemotactic response to ACC,but not of UW4 AcdS-and UW4 AcdS--G.Wheat seeds were soaked with 3 mM of ACC,arginine or succinic acid,and each seed was inoculated with 108 cfu of UW4-G or UW4 AcdS--G.After the seeds were pot-cultivated for 15 d,the seedling biomass and the number of rhizosphere colonization bacteria were determined.Compared with,the number of rhizosphere colonization bacteria and fluorescent bacteria of the treatment UW4-G with no chemoattractant were 178.63%(p<0.01)and 214.74%(p<0.01)higher than that of the control treatment(without chemoattractant and fluorescent labelled bacteria),while the,plant height and dry weight were 17.04%(p<0.01)and 27.85%(p<0.05)higher,respectively.Plant height,dry weight,and root dry height of the treatment with UW4-G with ACC were 23.11%(p<0.01),41.93%(p<0.01),and 127.58%(p<0.01)higher than that of control,respectively.Wheat biomass had no differences among the control and treatment with UW4-G with chemoattractant Arg or Suc.Sinilarly,wheat biomass had no differences among the control and treatment UW4 AcdS--G with Arg,Suc or ACC.The results showed that the rhizosphere colonization traits and plant growth promoting effect of Pseudomonas putida UW4 were closely related to the chemotaxis of ACC.