Identification And Colonization Of An Antagonistic Endophytic Burkholderia Cepacia Lu10-1 Isolated From Mulberry

Mulberry (Morus alba L.), an important substantial basis of sericultural industry, is widely planted about 7000 years'history in our country. In addition, it is also traditional officinal plant. However, it is always infected with many diseases in the past years, which has embarrassed severely the sustainable development of sericicultural industry. Presently pesticides application is the major messure of controlling the disease, but the use of pesticides is restricted because this may induce chemical residues in the environment. Compared with chemical control, biological control is more practical and safer in preventing the mulberry pathogens.Endophytic bacterum, which colonize in the host plant tissues without doing substantive harm to the host or gaining benefit other than securing residency, have successfully served as biocontrol agents in recent years. In addition, endophytic bacteria can exit in the plant well, and control disease stably. Consequently, endophytic bacteria are good biocontrol bacteria to control mulberry disease. However, little report could be found focusing the endophytic bacteria of mulberry.Lu10-1 strain, isolated from the healthy mulberry leaves, was an antagonistic bacterium that exhibited excellent resistant against various plant pathogen such as Ralstonia solanacearum and Colletotrichum morifolium hara. In this study, the final taxonomical status of strain Lu10-1 was confirmed to be a single species with the phenotypic and molecular methods for species identification. In addition, the population of strain Lu10-1 living in the mulberry tissues was analyzed, and the endophytic colonization rule of Lu10-1 was established with mulberry seedlings following different methods. The main results were showed as the followings:1. The result showed that strain Lu10-1 had broad antimicrobial spectrum by screening its antagonistic activity against Colletotrichum morifolium, Septogloeum mori, Pseudomonas syringae pv. Mori, and so on.2. Strain Lu10-1 was identified based on the analysis of its 16S rRNA gene sequence homology, the physiological and biochemical characteristics, and the recA gene sequence comparison. The results showed that Lu10-1 belonged to Burkholderia sp.. In the phylogenetic tree, Lu10-1 was the closest relative to Burkholderia cepacia (X80284) with more than 98% sequences similarity. The 16S rDNA sequences of Lu10-1 have been registered at GenBank database under the accession number EF546394. Finally, it was clearly demonstrated that the strain Lu10-1 fell into Burkholderia cepacia complex genomovarⅠby PCR with Bcc-specific and genomovar-specific primers based on recA gene. The final taxonomical status of strain Lu10-1 was confirmed to be a single species.3. Tagged with the drug resistance of Rif. (300μg/mL) and Amp.(300μg/mL), the strain Lu10-1 was inoculated by the way of stem and leaf acupuncturing, seed soaking, root soaking and leaf daubing. The population of strain Lu10-1 living in the mulberry tissues decreased as a whole after the treatment of seed dipping and root dipping. As for the population of strain Lu10-1 in mulberry leaves and stems after the treatments, it increased first then decreaed. Ultimately, it achieved a steady level in different mulberry tissues after 20 days. In the investigation of the colonization, it was proved that the strain Lu10-1 could colonize and transmit in the mulberry, and its resistance against plant pathogen did not change in the process of colonization compared to the original strains. Therefore, Burkholderia cepacia Lu10-1 would have a far future in the biological control of mulberry disease.4. The adsorption, multiplication and penetration of the Burkholderia cepacia Lu10-1 in the young roots of mulberry were examined with scanning electron microscopy at the different inoculation. The results showed as follows: The root hair surface and the region of elongation surface of mulberry were two preferential sites of adsorption and multiplication of the strain Lu10-1. The bacteria could enter the epidermal cells of mulberry roots by the lateral root junction, the root hair junction, the root hair surface, the cracks between epidermal cells, and epidermal cells of the young lateral root.5. In this study, we had maked the Burkholderia cepacia Lu10-1 with gfp gene. The results showed that the vector pGFP4412 produced high level of GFP, and the transformants appeared bright green under blue light. The esperiment of stability of the gfp-labeled strain indicated that its stability is very strong. Therefore, the strain of Lu10-1-gfp could statisfy the need of study in the colonization in the interior of the mulberry tissues.6. Microscopic observation of colonization of Burkholderia cepacia Lu10-1-gfp inside the mulberry tissues using confocal laser scanning microscope showed that the Burkholderia cepacia Lu10-1 could colonize and transmit in the mulberry. The bacterial cell infected mainly the inner cortex and vascular stele of mulberry root from the root hair, and the junction between primary and lateral root. By the vascular tissue, the bacteria migrated slowly from roots to stems and leaves. The bacteria were mainly located in the intercellular spaces and vascular tissue of mulberry. Otherwise, the strain Lu10-1 could not enter interior of the mulberry cells.