| Paenibacillus kribbensis PS04 is a biocontrol bacterium isolated from the insecticidal botanical garden of South China Agricultural University.The results of previous studies showed that PS04 had significant biocontrol effect on several fungal diseases such as rice sheath blight and rice blast.Its exopolysaccharides could induce many vegetables such as lettuce and swamp cabbage to develop resistance to the disease.And PS04 could promote the yield of rice to a certain extent.However,the mechanism of formation and action of PS04 biofilm has not been studied.This project mainly studied the effect of the morphological characteristics,components,colonization process,colonization of PS04 biofilm on induction of the disease resistance of rice,and the effects of rice root exudates on the formation of PS04 biofilm and its biological control function.The results were as follows:1.By measuring the growth curve of thallus,it was found that PS04 bacterial strain entered the logarithmic growth period after culturing for 12 hours and entered the plateau period after culturing for 36 hours,and the OD600 was stable at about 1.38.By measuring the growth curve of biofilm,it was found that PS04 biofilm began to produce after 24 hours,and the biofilm quantity tended to be stable after 72 hours.Through observation of PS04biofilm with scanning electron microscope,it was found that there was a little thallus adhered together after culturing for 24 hours,and after culturing for 72 hours the thalli were coated by exopolysaccharides,forming a stable reticular structure between the thallus,and the biofilm was mature.2.By using gas chromatography-mass spectrometry,it was found that the biofilm formed by PS04 after culturing for 24 hours was mainly composed of 37 substances including decamethylcyclopentasiloxane（16.80%）,2-nonone（8.96%）,dimethyl silane glycol（8.79%）,4-acetylbenzoic acid（7.19%）,and[（2,3-diphenylcyclopropyl）methyl]phenyl thioether（7.86%）.And the biofilm formed by PS04 after culturing for 72 hours was mainly composed of 20 substances including 4-acetylbenzoic acid（32.56%）,2,5-diketopiperazine（19.84%）,and of 2,3-butanediol（15.36%）.Through the reported literature review,2,3-butanediol was selected as bacteriostatic substance and verified by plate bacteriostatic experiment.It was found that the bacteriostatic rate of 250μmol/L2,3-butanediol to mycelium growth of rice sheath blight could reach 84.19%,which was of high bacteriostatic effect.3.The colonization process of PS04 on rice leaves was studied by using scanning electron microscopy（SEM）,and it was found that there was a little of bacterium secreted exopolysaccharides on the surface of rice after processing the rice leaves with PS04fermentation broth at 24 hours.After processing at 72 hours,the surfaces of rice leaves were coated by exopolysaccharides.Then in order to study the disease prevention effect of different colonization time,rice sheath blight was inoculated on rice leaves which processed with PS04 at 24 hours and 72 hours.It was found that the control effect reached94.79%and 94.87%respectively.Through quantitative real-time PCR of RNA sample extracted from rice leaves processed with PS04 at 24 hours and 72 hours,it was found that the expression of AOS gene in JA pathway was significantly up-regulated after 24 hours of PS04 colonization,but down-regulated after 72 hours of colonization.And the expression of LOX gene was not significant after 24 hours of colonization,and significantly down-regulated after 72 hours of colonization.In SA pathway,the expression of PAL gene was significantly up-regulated after 24 hours of colonization,but the difference between the expression after 72 hours of colonization and 0 hour of colonization was not significant.The expression of NAC gene after 24 hours of colonization was not significant,and significantly down-regulated after 72 hours of colonization.in ET pathway,the expression of EIN gene was not significant after 24 hours of colonization,and significantly down-regulated after 72 hours of colonization.The expression of PR3 gene related to disease course was not significant after 24 hours of colonization,and down-regulated after72 hours of colonization.And the expression of NPR1,PR5 and PR10 genes had no significant change after 24 hours and 72 hours of colonization.Through the enzyme activity assay of antioxidant,it was found that POD activity decreased by 21.95%after 24 hours of colonization,and increased by 164.88%after 72 hours of colonization;and SOD activity increased by 81.86%and 63.60%respectively after 24 hours and 72 hours of colonization;and CAT activity did not change significantly after 24 hours and 72 hours of colonization.4.Differentially expressed genes were studied by RNA-seq,and obtained 9422581 and8443150 reads respectively.There are 590 up-regulated genes and 469 down-regulated genes.Among the significantly up-regulated genes,the genes such as yes P,yes O,yyd M and mal L during biofilm formation were not reported yet.Among the significantly down-regulated genes,the genes such as tag U and ywq D during biofilm formation were not reported yet.These genes may be new genes related to biofilm formation.GO enrichment analysis showed that there were 451 GO terms in biological process,among which the metabolic process,cell process and mono biosynthesis were significantly enriched;and 289GO terms in molecular function,among which the catalytic activity,binding and transport activity were significantly enriched;and 72 GO terms in cell components,of which the membrane,membrane components and cell components were significantly enriched.KEGG enrichment analysis found that 540 genes were enriched in 21 categories,among which arginine biosynthesis,lysine degradation and benzoate degradation were related to the production of biofilm.Some biofilm-related genes were verified by quantitative real-time PCR,and the results showed that the expression of extracellular polysaccharide synthetic genes（spo0E,eps O,lev B）,2,3-butanediol synthetic genes（bdh A）and flagellin synthetic genes（flh F）were significantly up-regulated during the formation of biofilm,while the expression of late competence protein genes（com ER）and 2,3-butanediol synthetic genes（als E,als S）were down-regulated.5.In order to study the effect of rice root exudates on PS04 biofilm,we firstly measured the change of PS04 biofilm in LB medium with different amount of root exudates by crystal violet staining method.When 25μL,50μL,75μL and 100μL root exudates were added,the biomass of biofilm increased 1.53 times,1.87 times,2.33 times and 3.47times respectively.Then PS04 strain was cultured in LB medium with different amount of root exudates,and rice leaves in vitro was processed to compare the effect of root exudates on rice sheath blight.It was found that after adding 25μL,50μL and 100μL root exudates,the preventive effect of PS04 strain on rice sheath blight was increased by 2.08 times,2.10times and 2.99 times respectively,and the therapeutic effect was increased by 2.23 times,2.53 times and 3.81 times respectively.In conclusion,PS04 began to produce biofilm after growing for 24 hours,and the biofilm matured and produced 2,3-butanediol after 72 hours.PS04 initially completed colonization after processing the rice for 24 hours,and after 72 hours it produced mature biofilm and coated rice leaves.PS04 could improve the disease resistance of rice by inducing gene expression in SA pathway,SOD activity and POD activity in the process of colonization.Rice root exudates significantly promoted the formation and control of PS04biofilm.The transcriptome information of PS04 biofilm formation was obtained by transcriptome sequencing,and six genes that may be related to biofilm formation were screened out,which provided scientific basis for further study on the formation,colonization and gene function of P.kribbensis biofilm. |
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