Activity And Colonization Ability Of Beneficial Bacteria To Cropping Obstacless Of Rehmannia Glutinosa

Cropping obstacles of Rehmannia glutinosa is one of the main problems limiting the increasing of its production and it is a hot issue for traditional Chinese medicine to find out new ways to overcome the cropping obstacles of Rehmannia glutinosa at present. Former researches have indicated that the imbalance of Rhizosphere microflora is one of the main reasons. The main purpose of the study is using the helpful strains to overcome the cropping obstacles of Rehmannia glutinosa by studying thier biological activity and colonization capability in feilds.In the study, ten beneficial strains (strain 1-strain 10) were identified which had been isolated during the early study by observing their colony morphology, individual morphology as well as physiological and biochemical tests. Then the alignment and phylogenetic anaylysis of their 16S rDNA sequence were done by the software of DNASTAR and the Blast tool in the NCBI website. The results showed that the strains of strain 1,6 and 9 were Pseudomonas putida, strain 2 was Myroides odoratimimus, strain 3 was Bacillus subtilis, strain 4 was Acinetobacter sp., strain 5 was Psychrobacter alimentarius, strain 7 and strain 10 were Arthrobacter aurescens., and strain 8 was Proteus vulgaris.In order to study the preservation of the strains and meet the requirement of development, the best growth conditions for every strain were explored respectively, including the most appropriate medium, pH value, temperature, rotation speed and preservation conditions. The results suggested the optimum media were LB (for strain 2,3,5,7,9 and 10) and NB (for strain 1,4,6 and 8) respectively; the optimum pH values were pH 7 (for strain 1,3,4 and 10) and pH8 (for strain 2,5,6,7,8 and 9) respectively; the optimum temperature wre 28℃(for strain 1,2,3,6,8,9 and 10),30℃(for strain 4 and 7), and 25℃(for strain 5) respectively. The strains which are suitable for preserving by slant passage at 28℃are 1,3,6; 2,7,9 and 10 are suitable for preserving by slant at 4℃; 1,3,4,5,6,7,8,9 and 10 are suitable for preserving in the liquid glycerin; 2 is suitable for preserving by puncture; 2,4,5,8 are suitable for preserving by sand tubes; 1,3,4,5,6,7,8,9 and 10 are suitable for preserving by ultra-low temperature freezing.After the addition of Rehmannia glutinosa extract into MS medium, the plantlets of Rehmannia glutinosa were died. The Rehmannia glutinosa tissue culture plantlets which were dealt by Rehmannia glutinosa extract survived with their roots being immersed into 1-10 strains, but the effects of different strains were different. The effect of strain 9 was the most obvious. So, strain 9 was selected for the further research. According to the characteristics of Pseudomonas, three parents hybridization test were used to mark strain 9 with Lux light gene. The strain which was successfully marked was named C-9. Comparing of the physiological and biochemical characteristics and the effects of strain 9 and C-9 showed there was no obvious difference between the both strains (P>0.05).Applying the C-9 into the roots of Rehmannia glutinosa sterile culture and culturing the plantlets in sterile condition, then the soil around the root were sampled and observed the colonization of C-9 every five days. The strains could be detected in 95 days. Applying the C-9 into the field, the strains could be detected in 55 days. The strains which were applied into the soil around the roots of Rehmannia glutinosa could affect the Microflora in the soil. During the early stage, the numbers of bacteria and fungi increased obviously (P<0.05) while the number of actinomycetes decreased. During the late stage, with the amount of C-9 decreasing, the numbers of bacteria and fungi were decreasing while the number of actinomycetes was rising. When the C-9 became less and less, the number of each kind of microorganism became more and more till approaching to the levle before the C-9 was applied into the field. So it proved that the strain C-9 had affected the Microflora in the soil around the roots of Rehmannia glutinosa obviously (P＜0.05).