Research Of Interaction Between Plant Roots And Rhizosphere Beneficial Bacillus Strains N11and SQR9

The control of Fusarium wilt is a world wide problem. The traditionally farming control methods have rarely provided long-term control in any production area, while selection of resistant cultivars and chemical application are not effective enough for control this disease, and also the latter causes environmental problems. Recently, biological control of soil-born disease is an efficient and environmentally friendly way and is receiving increased attention.In this study, the capability of the specific bio-organic fertilizers made of organic fertilizer (pig manure compost and amino acid fertilizer) fermented with antagonisms Bacillus amyloliquefaciens N6or/and Bacillus subtilis N11, as well as the normal BIO developed before in this laboratory, to control the Fusarium wilt of banana was evaluated in pot experiments. The colonization pattern of GFP tagged N11and SQR9, another biocontrol agent isolated from the cucumber rhizosphere and being shown a very broad antagonistic spectrum to Fusarium oxysporum, on banana roots was determined in hydroponic, sand and natural soil system, respectively. The appearance of the biofilm formation of the two Bacillus strains in vitro, the method for quantitative measurement of the biofilm biomass, and the dynamics of the biofilm biomass were studied. The response of N6, N11and SQR9towards the root exudates of banana and cucumber, as well as the organic acids composition, was investigated. Subsequently, the transcriptional profiling of SQR9towards different root exudates were analysed by Solexa high-throughout transcriptome sequence. At last, the effect of cheA deletion on biofilm formation of SQR9was studied. The main results obtained are listed as follows.1. The capability of the specific bio-organic fertilizers (BIO2-4) for banana plants made of Bacillus amyloliquefaciens N6or/and Bacillus subtilis N11isolated from the banana rhizosphere to control the Fusarium wilt of banana was evaluated in pot experiments, and the normal bio-organic fertilizer (BIO1) made of Paenibacillus polymyxa SQR21and Trichoderma harzianum SQR-T37was also used for comparison. Results indicated that the application of BIO could decrease the disease incidence (DI) of banana from93.3%to below33.3%, and the DI in treatments applied with specific BIO (BIO2-4) was even lower than16.7%. The F. oxysporum was recovered from the bulk and rhizosphere soil by Quantitative Real-time PCR. Results revealed that the copy numbers of ITS gene of FOC in bulk soil were similar among all treatments. However, application of BIO could decrease the copy numbers of ITS gene of FOC inrhizophere soil from1.59×105g-1soil (control) to less than6×104g-1soil, and the treatments with specific BIOs (BIO2-4, T3-5) even suppressed the copy numbers to below1.5×104g"1soil. The microbial communities in the banana rhizosphere in all the treatments were analyzed by PCR-DGGE. No significant dissimilarities in species composition of the fungal communities were found. However, application of specific BIOs (T3-5) significantly increased the bacterial species.2. An E. coli-Bacillus shuttle vector pHAPII, which contained a continually expressed Green Fluorescent Protein (GFP), was attempted introduce into N11by electroporation to obtain the GFP-tagged N11(N11-gfp). The cells and colonies of N11-gfp could emit bright green fluorescent under the microcopy. The growth speed and the antagonistic ability against FOC of N11-gfp were identical with that of the original strain. Later, the distribution of N11-gfp and SQR9-gfp, constructed by this laboratory previously, in banana rhizosphere was determined in hydroponic, sand and natural soil system by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), respectively. Results indicated the colonization patterns of the biocontrol agents were similar among the three systems. Micrographs showed that2to4days after inoculation, the antagonists formed microcolonies and biofilm on the roots surface of banana plants, and the colonization mainly occurred on the elongation and differentiation zones of the main roots, the lateral roots, the junctions between roots, as well as the root hairs, while very few cells attached along the root tip. The quantitative analysis revealed that4days after inoculation, the population of N11-gfp and SQR9-gfp maintained at about105-106CFU·g-1roots, and106-107CFU·g-1soil.3. The biofilm formation of several Bacillus strains in microtitre was studied in vitro. The biofilm formed by B. subtilis168in both LB and MSgg medium was thick, while the wide type strains FZB42, N6, N11and SQR9could form thin biofilm in both medium. It was found the biofilm formed in MSgg was thinner, also with more complex structure. The florescent microscopy pictures revealed that the biofilm of N11-gfp and SQR9-gfp formed on cover slides and microtitre presented complex three-dimensional architecture. The CLSM graphs of biofilm formed by SQR9-gfp confirmed this phenomenon. The traditionary violent staining method and a vortex method for quantitative measurement of the biofilm biomass were found to be identical, with a correlation index R2of0.9313. The biofilm formed by Bacillus strains displayed some edge-effects in the microtitre. At last, the dynamics of the biomass of biofilm and suspended cells of the four wide type strains were monitored. The biofilm biomass of each strain continuously increased from24h after inoculation, and maintained at about60h post inoculation. At this time the biomass of suspend cells began to increase. The disassembly of the biofilm occurred at96h after inoculation. The biofilm biomass of all the four strains in MSgg were higher than those in LB, and the biomass of the biofilm formed by FZB42and SQR9in both of the medium were larger than N6and N11.4. The colonization of N11-gfp (isolated from banana rhizosphere) and SQK9-gfp (isolated from cucumber rhizosphere) on their original host plants was more effective than on the other plant. N6and N11displayed strong chemotactic reaction towards both the root exudates of banana and cucumber. SQR9showed ordinary reaction to the two kinds of root exudates. The root exudates of banana could significantly stimulated the biofilm formation of N6and N11, while both the two kinds of root exudates could significantly induced the biofilm formation of SQR9. High performance liquid chromatography (HPLC) analysis of the root exudates of banana revealed the existence of oxalic acid, malic acid, and fumaric acid, while oxalic acid, malic acid, and citric acid in those of cucumber. N6and N11showed intense chemotaxis reaction to malic acid, citric acid, and fumaric acid, while only the first two organic acids could significantly attract SQR9. Fumaric could significantly stimulate the biofilm formation of all the three strains, while citric acid could induce the biofilm formation of N6and SQR9. Colonization assay indicated that fumaric acid could improve the colonization of N11-gfp on banana and cucumber; while malic acid, citric acid, and fumaric could induce the survival of SQR9-gfp on banana or/and cucumber.5. The shuttle vector pXKZ, which contained a competence factor ComK under the control of a xylose induced promoter (PxylA), was introduced into wide type Bacillus strains SQR9by conjunction. It was found that transformation of9kz by different plasmids was available with induction of xylose, and the transformation efficiency ranged from103to10CFU·μg-1. The transformation condition was optimized by Box-Behnken experiment with Response Surface Methodology (RSM), and the best situation was found to be:induction with0.2%of xylose for1h when the OD600of the suspension reached0.5, the transformation efficiency was approximately10CFU·μg-1DNA. The fragments contained the upstream and downstream arm for recombination with cheA, the suicide vector constructed by this fragment and pMD19-T, and the multimerzation of this suicide plasmid, were introduced into SQR9kz by induced transformation, respectively. Several corrected mutants were obtained by confirmation with PCR, and the highest efficiency were obtained for multimer transformation, which was still lower than10CFU·μg-1DNA. Then the pXKZ were eliminated by culture the mutant for several generations. The chemotactic response of the cheA null mutant towards citric acid was abolished. Its growth speed was similar with the wide type strain, while the biofilm formation delayed. The transmission electron microscopy (TEM) graphs revealed that the mutant failed to form flagellum, while the flagellums were found around the wide type cells. We speculated that the knockout of cheA caused some polar effects on the sigD in the downstream, thus influence the formation of flagellum, and disturb the chemotactic reaction, as well as delay of the biofilm formation, which needs complementation of cheA and sigD for confirmation.6. The concentrated root exudates of cucumber, maize and banana could all significantly stimulate the growth of SQR9in1/2MSgg medium. Root exudates of maize could induce the biofilm formation of SQR9in1/2MSgg medium. The transcriptional profiling of SQR9towards the three kinds of root exudates under shaken condition, as well as its respond to maize exudates under static condition, were analysed by Solexa high-throughout transcriptome sequence. Results indicated the main regulated genes belonged to metabolism (mainly regarding carbohydrate, amino acids, lipid and nucleotide), protein translation and folding, membrane transport (ABC transporters), signal transduction (two-component system) and cell chemotaxis and motility. Some differences exist within the genes mediated by different root exudates. In static condition, the transcriptome of SQR9respond to maize roots after incubation for24h and48h, were quite different.