| In the 21st century,excessive application of chemical fertilizers and pestici-des has seriously damaged land resources and brought a series of food safety problems.Plant growth-promoting bacterial fertilizer refers to biological agents made by plant growth-promoting rhizobacteria(PGPR).PGPR mainly includes nitrogen-fixing rhizobium,phosphorous or potassium-dissolving bacteria,and other biocontrol bacteria,etc.They can colonize and survive in the root system or rhizosphere soil of crops,thus promoting the growth and development of crops or inhibiting the growth of pathogens in the surrounding environment.As a kind of microbial fertilizer,PGPR has a broad application prospect in developing green agriculture.However,The effect of PGPR in promoting growth is not stable in practical application.Though its growth-promoting effect is outstanding in the laboratory,massive field application is unstable due to geographical location,soil nutrition,crop varieties and year of application.Therefore,to carry out a research on efficient strain breeding and growth-promoting mechanism of PGPR is of great significance.Whether PGPR can play an effective role mainly depends on whether it can win in the competition with other native microbial communities in the rhizosphere of plant,so as to grow and reproduce stably in the root or rhizosphere soil of it.PGPR can sense the changes in the concentration of root exudates in the rhizosphere environment of plants,reach the rhizosphere through chemotaxis,and form a stable bacterial membrane structure in the rhizosphere.Chemotaxis is the reaction of bacteria sensing the concentration gradient of chemicals in the environment,to attract attractants or stay away from repellents with the help of moving organs such as flagella.After the gene related to chemotaxis was knocked out,the ability of survival and reproduction of PGPR in plant rhizosphere decreased significantly.It was generally believed that the colonization quantity of PGPR in plant rhizosphere was positively correlated with its growth-promoting effect.In this study,we tried to find out the key chemoattractant that promote PGPR to colonize the rhizosphere of crops,and carried out the selection and breeding of high-efficiency PGPR strains to stabilize and improve the field application effect of PGPR.The main research results are as follows:1.ACC is a strong chemoattractant of Pseudomonas putida UW4Comparing the chemotaxis response intensities of UW4 to ACC and the other 8 amino acids and 7 organic acids commonly found in plant root exudates by plate droplet chemotactic analysis,the author found that arginine and succinic acid were strong chemoattractant to UW4,while the chemotactic strength of UW4 to ACC was significantly higher than them.Capillary chemotaxis method was used to quantitatively determine the chemotaxis threshold and peak concentration of UW4 to ACC,arginine and succinic acid.The results showed that the chemotaxis threshold and peak concentration of UW4 to ACC were 5×10-5 M and 5×10-2 M respectively and its chemotaxis response intensity was higher than that of arginine and succinic acid.Furthermore,the chemotaxis response strength of UW4 to ACC and artificial root exudate(ARE)was compared by quantitative capillary chemotaxis method.The results showed that:UW4 had the strongest chemotaxis response on ACC,weaker on ACC and ARE mixture,and weakest on ARE,indicating that ACC was a strong chemoattractant of UW4.2.ACC should be the key chemoattractant for Pseudomonas putida UW4 to colonize at plant rhizosphere and fungal hyphaeCo-culture of UW4,UW4△AcdS and UW4△cheR and complement strain UW4△cheR+cheR with Agaricus bisporus indicated that the colonization of UW4 and complementary strain UW4△cheR+cheR at fungal hyphae was significantly higher than that of AcdS gene knockout strains UW4△AcdS and cheR gene knockout strains UW4△cheR(p<0.05)Agaricus bisporus has the same ethylene synthesis pathway as plants,and the mycelium secretion also contains ACC.The above strains were inoculated into wheat seeds for pot experiment and the results showed that the colonization of different strains in wheat rhizosphere was similar to that of Agaricus bisporus.The wheat root length,root weight,stem length and stem weight treated by UW4 and UW4△cheR+cheR were significantly higher than that of UW4△AcdS and UW4△cheR(p<0.05),indicated that ACC should be the key chemoattractant of UW4 toward the plant rhizosphere and fungal hyphae to colonize.3.Increase the metabolic rate of Pseudomonas putida UW4 to ACC can enhance its chemotaxis response intensity and quantity of rhizosphere colonizationWhether increasing the metabolic rate of metabolically dependent chemoattractant can enhance the response chemotaxis strength of bacteria has not been reported.The author used homologous recombination technology to knock the PB20 sequence of bacterial constitutive strong promoter into the upstream of UW4 AcdS gene without trace,and successfully constructed the ACC deaminase high-activity strain UW4-PBA.The expression of AcdS gene in UW4-PBA strain was measured by fluorescence quantitative PCR,which was about 30 times that of the UW4 without ACC induction,and increased to about 81 times after induction with ACC for 45min,while the AcdS expression was no significant difference between UW4 and UW4-PBA after ACC induction(p<0.05).In addition,the ACC deaminase activity of UW4-PBA strain was about 6μmoL/μg·min higher than that of UW4,and the datas differed significantly(p<0.05).The results of qualitative chemotaxis showed that the diameter of the chemotaxis circle of UW4-PBA to ACC,gamma-aminobutyric acid,arginine and succinic acid was significantly larger than that of UW4,and there were significant differences between datas(p<0.05).Inoculating UW4△AcdS+PB20-AcdS,UW4△AcdS+PB10-AcdS,UW4△AcdS+PB1-AcdS,UW4△AcdS+PA-AcdS strains which with strong,medium and weak promoters PB20,PB10,PB1 and AcdS gene self promoter constructed by a member of our research group,wild strain UW4 and UW4△AcdS into the wheat rhizosphere,the researcher through aseptic bottle culture experiment found that the quantity of UW4-PBA strains colonized in wheat rhizosphere was the largest,while that of UW4△AcdS+PB1-AcdS and UW4△AcdS were comparatively small,and there were significant differences between datas(p<0.05).Planting the wheat seeds into pots(non sterile conditions)after inoculating the bacterial strain,the author found that:the number of UW4-PBA strains colonized in potted wheat rhizosphere was significantly higher than the other six strains(p<0.05),UW4△AcdS+PB20-AcdS and UW4-PBA treated wheat root length was significantly higher than that of other strains,the root weight of wheat treated with UW4△AcdS+PB20-AcdS,UW4-PBA and UW4 strains was significantly higher than that of other strains.The stem weight of wheat treated with UW4△AcdS+PB20-AcdS,UW4-PBA strains was also significantly higher than that of other strains(p<0.05).In conclusion,improving the metabolic rate of ACC can significantly enhance the chemotaxis response intensity of UW4 to ACC and its chemotaxis to rhizosphere,thus,the research confirmed that ACC is the key chemoattractant for the chemotaxis colonization of UW4 at the rhizosphere of plants.4.Identification of UW4 ACC chemoreceptor of Pseudomonas putida UW4The expression of 28 hypothetic chemoreceptor proteins of UW4 was measured through fluorescence quantitative PCR,the four candidate chemoreceptor proteins with high expression under ACC induction were wp116,wp375,wp616 and wp718.Furthermore,the knockout mutant and complement strain of these four chemoreceptor genes were constructed.The chemotaxis experiment on the flat plate drip showed that UW4△wp116completely lost chemotaxis to ACC,gamma-aminobutyric acid and arginine chemotaxis,but its chemotaxis to succinic acid was unaffected compared with UW4,UW4△wp116+wp116 restored chemotaxis to ACC,gamma-aminobutyric acid and arginine the chemotaxis strength of UW4△wp375,UW4△wp616 and UW4△wp718 to ACC,gamma-aminobutyric acid and arginine was slightly weaker compared with UW4 Therefore,it was deduced that wp116 may be the chemoreceptor of ACC,but not a specific receptor,and chemoreceptor of ACC and some protein amino acids may be wp116.The ligand-binding domain of wp116 was expressed in Escherichia coli and purified.The affinity of ligand-binding domain of wp116 to ACC was analysisd by fluorescence thermal drift,the results showed that the ligand-binding domain of wp116 had a strongest affinity with ACC.Accordingly,it was speculated that wp116 should be the chemoreceptor of ACC.1-aminocyclopropane-1-carboxylate(ACC)is a common plant root exudates but has not been reported as a chemoattractant of bacteria.We found that UW4 can utilize ACC,whereas its ACC deaminase(ACC deaminase,AcdS)gene deletion mutant strains UW4△AcdS and chemotactic protein methyltransferase(chemotaxis protein methyltransfe-rase,CheR)gene deletion mutant strains UW4△cheR did not utilize ACC.Compared with other chemoattractants,UW4 has the strongest chemotactic response to ACC,and increasing the metabolic rate of Pseudomonas putida UW4 to ACC can enhance its chemotaxis response intensity and quantity of rhizosphere colonization.Thus,it can be speculated that ACC is a new metabolics-dependent chemoattractant of UW4 and the key chemoattractant for Pseudomonas putida UW4 colonization at the rhizosphere. |
