Screening Of Rhizospheric Specific Microbe And Its Toxic Action On Continuous Cropping Radix Pseudostellariae

The study found that the traditionally medical plants in China,such as Radix Pseudostellariae,Radix Notoginseng,Radix Rehmanniae ect.,continued to crop,which resulted in declining in the production and reducating in the quality.The previous study found that the rhizosphere of the plant have many quorum-sensing microorganisms.In this experiment,we saw Radix Pseudostellariae rhizospherical soil bacteria as the research objects.For the view of the quorum-sensing,we studied how bacteria associated with Radix Pseudostellariae rhizosphere impacted Radix Pseudostellariae.The detail work was introducted as following:1 Flat-panel painting parallel lines screen quorum-sensing bacteria.According to the principle of the the Chromobacterler violaceum showing purple,flat-panel painting parallel lines method was used for screening quorum-sensing bacteria:the control group of five,replanting treatment 12,continuous cropping 15.Through 16SrDNA molecular identification,these groups mainly belonged to Serratia.2 96-wells liquid culture screen quorum-quenching bacteria.According to the principle of the the Chromobacterler violaceum showing purple,96-wells liquid culture method was used for screening quorum quenching bacteria:the control group of five,replanting treatment one,continuous cropping eight.Through 16SrDNA molecular identification,T144 strain belonged to the Bacillus thuringiensis;T232 strain belonged to the Pseudomonas,other quorum quenching bacterium were Serratia.3 The construction of Radix Pseudostellariaetissue culture system and impactation of rhizosphere bacteria associated with Radix Pseudostellariae to the Radix Pseudostellari-ae tissue culture.The system of the medical Radix Pseudostellariae tissue culture was constructed.According to the different growth stages of the Radix Pseudostellariaeneeding the diefferent nutritions,we optimizated the hormone types and concentrations 0.05mg/L NAA(1-naphthlcetic acid)+3.0mg/L ZT(6-(4-Hydroxy-3-methylbut-2-enylamino)purine)+MS was the best medium for primary culture;0.05mg/L NAA(1-naphthlcetic acid)+2.5mg/L 6-BA(6-benzy lam in op-urine)+MS was the best medium for secondary culture.We respectively put the quorum-sensing strain Serratia T246 and quorum-quenching strain Bacillus thuringiensis T144 into the Radix Pseudostellariae tissue culture,we found Serratia T246 would kill the Radix Pseudostellariae tissue culture;Serratia T246 was harmful for the Radix Pseudostellariae.The diluted Bacillus thuringiensis T144 had no obvious effect on the Radix Pseudostellariae.The next moment,we would mixed T246:T144 by 1:1,2:3,3:7 ratioes,respectively.In the conditions of 25 degrees and 1000-15001x,They were inoculated in the Radix Pseudostellariae approximate 13 days.The Radix Pseudostellariae had no obvious symptoms.4 Construction of fluorescent protein engineering bacterium in rhizosphere of the Radix PseudostellariaeThe pPBUD-cat was obtained by inserting the cat into the amp site in the pPBUD.The pPBUD-cat-egfp,which was acquired by plugging gfp in the plasposon of the pPBUD-cat,transferred from E.coli DH 5a to Serratia T246 through electroporation.Clones expressing EGFP was screened out under a fluorescent microscope,it was proved that pPBUD-cat-egfp in these clones by PCR.We expected to observate real-timely,in situ bacteria in Radix Pseudostellariaevivo or in rhizosphere colonization,distribution through Serratia T246-GFP.The method is helpful to learn the relationships of both host plants and bacteria in Radix Pseudostellariae vivo or in rhizosphere.