Directed Screening Of Endophytic Bacteria With New QS Quenching Enzyme Gene From Pogostemon Cablin And Their Effects On Ralstonia Solanacearum Wilt

Pogostemon cablin(Blanco)Benth leaves,stems can be used as medicine,which has the effects of aromatizing dampness,harmonizing stomach and relieving vomiting,removing heat and relieving exterior syndrome.Pogostemon cablin has become a genuine medicinal material in Guangdong,one of the famous “top ten southern medicines”,and one of the varieties with the largest market demand,but the resources are scarce.Ralstonia solanacearum wilt is the most serious systemic disease in Pogostemon cablin cultivation and serious threat to the survival and development of Pogostemon cablin industry.At present,there is no effective prevention and control method for R.solanacearum wilt,so it is urgent to develop new prevention and control methods.Previous studies have shown that the pathogenic process of R.solanacearum is mainly regulated by the quorum sensing(QS)system of new signaling moleculesmethyl 3-hydroxypalmitate(3-OH PAME)or methyl 3-hydroxymyristate(3-OH MAME),rather than the general molecular acylated homoserine lactones(AHLs).It has been found that endophytic bacteria can stably coexist with plants and have the ability to promote growth and disease resistance,but the study of endophytic bacteria with novel QS quenching enzyme in the biocontrol R.solanacearum wilt of Pogostemon cablin has not been reported.In this study,the high-throughput sequencing was used to analyze the taxa of endophytic bacteria in Pogostemon cablin to obtain the dominant flora information,and the dominant strains were directed isolation by a special selective enrichment culture.Endophytic bacteria with both QS quenching enzyme gene and antagonistic activity were screened from dominant strains,their disease resistance and biocontrol effect in R.solanacearum wilt of Pogostemon cablin were analyzed by pot experiments,and their resistance mechanisms were elucidated.The implementation of this project will provides a basis for the establishment of a new type of R.solanacearum wilt biocontrol system.It is important to promote Pogostemon cablin planting,solve the shortage of resources and develope Pogostemon cablin industry.The specific results of this thesis are as follows:1.The results of 16 S r DNA high throughput analysis showed that the total number of Operational Taxonomic Units(OTUs)in roots,stems and leaves of Pogostemon cablin were 4249,1577 and 1562,respectively.The unique OTUs in roots,stems and leaves were 3170,582 and 1096,respectively.The OTUs shared by roots and stems were 930,the OTUs shared by roots and leaves were 365,the OTUs shared by stems and leaves were 281,and the OTUs shared by roots,stems and leaves were 216.The diversity of endophytic bacterial communities in roots of Pogostemon cablin was the highest(Chao1 index,ACE index),followed by leaves and stems.The main groups of endophytic bacteria in the roots and stems of Pogostemon cablin were very similar at the levels of phylum(Proteobacteria),class(Alphaproteobacteria and Gammaproteobacteria),order(Pseudomonadales),family(Pseudomonadaceae)and genus(Pseudomonas).The dominant endophytic bacteria in leaves were the same as those in roots and stems at the phylum and class levels,but there were significant differences in dominant groups at the order,family and genus levels.The dominant orders in leaves were Rhizobiales(14.61%),family [Moraxellaceae(4.78%)and Sphingomonadaceae(4.42%)],genus(Methylobacterium,9.58%).2.40 strains of dominated endophytic bacteria were isolated from Pogostemon cablin by Pseudomonas sp.enrichment medium.Among them,16 strains(40.00%)contained 3-OH PAME quenching enzyme gene,5 strains(Pseudomonas sp.02,Pseudomonas sp.09,Pseudomonas sp.11,Pseudomonas sp.26 and Pseudomonas sp.31)have obvious inhibitory effect on R.solanacearum,4 strains(Pseudomonas sp.02,Pseudomonas sp.09,Pseudomonas sp.26 and Pseudomonas sp.31)had both 3-OH PAME quenching enzyme gene and R.solanacearum antagonistic activity.The analysis of the plant growth-promoting characteristics of 40 Pseudomonas sp.showed that: strains with the activities of secreting indole acetic acid(IAA),siderophore,protease,organic acid and 1-amino-cyclopropane-1-carboxylie acid(ACC)deaminase,nitrogen fixation,dissolving organic phosphorus and inorganic phosphorus,and potassium dissolution accounted70.00%,47.50%,95.00%,57.50%,42.50%,100%,97.50%,92.50%,52.50%,respectively.3.Under the infection condition of R.solanacearum,the inoculation of5 endophytic bacteria strains(Pseudomonas sp.02,Pseudomonas sp.09,Pseudomonas sp.11,Pseudomonas sp.26 and Pseudomonas sp.31)with antagonistic activity could significantly improve the fresh weight,dry weight,leaf numbers,plant height and chlorophyll level of Pogostemon cablin,showing a good promotion effect.Among them,Pseudomonas sp.31 had the best growth-promoting effect.Compared with the control,fresh weight,dry weight,leaf numbers,plant height and total chlorophyll content of Pogostemon cablin inoculated with Pseudomonas sp.31 increased by1157%,25%,1845%,1560% and 29%,respectively.The inoculated endophytic bacteria strains(Pseudomonas sp.02,Pseudomonas sp.09,Pseudomonas sp.11,Pseudomonas sp.26 and Pseudomonas sp.31)compared with the control group without endophytes,the disease incidence of R.solanacearum wilt of Pogostemon cablin decreased by 50.00%,50.00%,18.75%,43.75% and 62.50%,respectively;the disease index decreased by 57.81%,62.50%,18.75%,54.68% and 70.31%,respectively.The growth-promoting and disease-resistant effects of endophytic bacteria(Pseudomonas sp.02,Pseudomonas sp.09,Pseudomonas sp.26 and Pseudomonas sp.31)carrying novel QS-quenching enzyme genes were better than those of Pseudomonas strains(Pseudomonas sp.11)without QSquenching enzyme genes.4.The 3-OH PAME quenching enzyme gene of Pseudomonas sp.31 was directed evolution in vitro,and a broad-spectrum quenching enzyme with co-degradation activity of 3-OH PAME and 3-OH MAME was obtained.The degradation rates of 3-OH PAME and 3-OH MAME were 19.84% and82.27%,respectively.The novel broad-spectrum QS quenching enzyme could significantly reduce the athletic ability(athletic ability decreased by34.62%)and extracellular polysaccharide secretion of R.solanacearum,inhibit R.solanacearum growth(growth rate decreased by 56.25%)and biofilm formation(decreased by 73.53%)in vitro.The results showed that endophytic bacteria with both novel QSquenching enzyme gene and R.solanacearum inhibitory activity had good plant growth promoting effect and the disease resistance of Pogostemon cablin to R.solanacearum wilt.They provide a new method for the prevention and control of R.solanacearum wilt of Pogostemon cablin,and are of great significance for solving the shortage of Pogostemon cablin resources and developing Pogostemon cablin industry.