| More than 250 plant diseases caused by Gram-(Gram-negative) bacteria lead to a serious reduction of crops of plant and economic losses. Of all the agricultural diseases that threaten citrus crops, citrus bacterial canker disease(CBCD) is one of the most serious plant diseases which caused by the bacterium Xanthomonas citri subsp. citri(Xcc). CBCD distribute in more than 30 countries including China and lead to the serious lost of citriculture. In the affected areas, the usage of cupric products and agricultural antibiotics are traditional control methods against CBCD, while they are not effective. On the contrary, these methods result in the eruption of red spider and the over standard rate of antibiotics and heavy metal ions. The seriously infected plants have to be burnt to stop the spread of CBCD. Bacillus as a kind of commonly used biocontrol strains have good prevention and cure effect on plant pathogenic bacteria and CBCD. The study of antimicrobial genes and antimicrobial material screening is helpful for the description of the biocontrol mechanism and the applications of high efficient biocontrol strain.In this study, The Xanthomonas citri subsp. citri(Xcc) and Bacillus subtilis CQBA03 were used as the research materials. The genomic library of Bacillus subtilis CQBA03 was constructed by using the method of constructing a Fosmid library. Pathogenic bacteria of CBCD Clones were obtained through the following steps: library induction, directed screening of the library. By the process of cloning, sequencing, analysis, bioinformatics analysis, expression and purification of antimicrobial genes of the bacterial pathogens, the metabolism pathway of antibacterial material related to this strain was revealed and a new secreted antibacterial protein was obtained as well.The deformation and lethal effect of the secreted D-amino acids from Bacillus amyloliquefaciens CQBA03 strains on Xcc was studied. Effect of D type amino acid on the prevention and treatment of CBCD was also carried out under laboratory condition. This study will provide theoretical basis for revealing the mechanism of the antibacterial mechanism of Bacillus amyloliquefaciens CQBA03 strain and the application of this strain.①The construction of genomic fosmid library of Bacillus amuefaciens CQBA03 strainA genomic fosmid library of CQBA03 was constructed and 2, 3000 clones was obtained.The size of the library is 920 Mb, about 244-263 times as much as the genome of Bacillus amuefaciens CQBA03(256 times on average).Inserted fragment size is from 36 kbp to 50kb(about 40 kb on average). Among the inserted fragment, the size of about 40.8% is between 31 kb and 35 kb, about 36.0% is between 35 kb and 40 kb. The no-load rate was lower than 1.5%。② The obtain and analysis of anti CBCD clones1)The strains were cultured on plate screening culture medium at 4℃. From 1, 0000 library clones, 66 clones with Secreted protease were obtained. DNA of one clone with the strongest exocrine protease function against CBCD was sequenced.2)A 58785 bp length sequence with 49.13% of GC was obtained by sequencing 454.By bioinformatics analysis, we infer the sequence contain 344 open reading frames. After comparison, 75 ORFs were similar to the known genes. Among the ORFs, 25 genes were related to anti microbic, 26 genes were related to physiological and biochemical enzymes, one gene was related to insecticidal toxin domain protein, and the remained 23 genes were related to proteins of unknown function. 24 related complete metabolic pathways were obtained by the analysis of metabolic pathway. The metabolic pathways related to anti microbic contain degradation and synthesis of ketone bodies. Biosynthesis pathway and of 12-14 and 16-membered macrolides were obtained from the genome of CQBA03.③ Obtaining, expression and purification of antibacterial geneAfter sequence analysis, 7 genes that might have antibacterial function were screened out. The genes were cloned by PCR and connected to PMD19 T vector, and then their function of anti CBCD was tested. The anti CBCD Fosmid peptide gene of 132bp(Accession number Bank It1690482) obtained encodes a small 43 AA peptide. The signal peptide site was 27-28 AA, molecular weight is 4772 Da, p I is 6.38. By comparison, the gene is highly homologous with the condensation zone of linear gramicidin enzyme. This gene has not yet been reported, so we inform it is a new functional gene.By constructing prokaryotic expression vector, induced expression and purification, fusion protein of 21 KD with HIS-tag tag was successfully obtained. However, the fusion protein obtained lost its antibacterial function.4. Discovery of antibacterial action of D-amino acids④ The discovery of antibacterial effect of D-amino acidsBy analysis of the content of D type amino acids in exocrine liquid culture of Bacillus amyloliquefaciens strain CQBA03.1)By early speculation and analysis, we analyze understand the content of D type amino acids in liquid culture of Bacillus amyloliquefaciens strain CQBA03 exocrine, we found that CQBA03 strain secrete low levels of D-amino acid(D-aa) that can lead to obvious morphological change and abnormal division of pathogenic bacteria of CBCD.2)However, when the D-Leu concentration in the LB liquid medium increased to 7 m M, the morphology of Xcc changed. Four distinct morphological periods existed①Bacteria size increase phase. ②Chain-like bacteria phase.③Protoplasmic expansion phase.④Cell wall phase.3)We stained the different culturing stages of Xcc101 treated with 7 m M D-Leu using the LIVE/DEAD Bac Light Bacterial Viability Kit. After being treated for 1 h, a large number of red-labeled dead bacteria were observed illustrating the acute death of Xcc. After being treated for 6 h, the bacteria turned into strong active chains that were green-labeled under fluorescence microscopy. After 10 h treatment, the bacteria protoplasm did not expand. Large amounts of red-labeled dead bacteria were observed, indicating that the bacterial morphology had not changed, but that the cell membrane was damaged. Nearly all the bacteria observed in the control group that was not treated by D-leu fluoresced a vibrant green at each culture phase.4)The determinations were repeated three times using eight replicate wells per run. The D-Val MIC for Xcc was highest at 18 m M, followed by D-Phe at 15 m M and D-Leu at 7 m M. In contrast, 50 m M L-Leu, L-Phe, and L-Val had no impact on the growth or morphology of Xcc. These results show that D-aas can inhibit Xcc101 growth. Therefore D-leu was selected for further studies, due to its high toxicity to Xcc.5)Within the peptidoglycan extracted from the Xcc, the amount of Arg, Ser, Leu, Ala, and Cys changed significantly(over 17%) following D-Leu treatment, whilst the concentration of other amino acids did not change significantly(less than 5%), in comparison with the control group. The content ratios of Arg, Ser, Ala, Leu and Cys6) Preventive effects of D-Leu on CBCD under laboratory conditionsThe addition of 7 m M D-Leu could lead to significant morphological changes in the Gram- plant pathogenic Xcc101 and eventually death. A variety of other D-aas may also have similar antibacterial effects on other plant pathogenic bacteria. Our results show that, under laboratory conditions, D-Leu has a significant effect on the control of CBCD. Consequently, we suggest that spraying D-Leu directly onto the plant would be an effective method for destroying harmful bacteria. Alternatively or additionally, the addition of D-Leu to botanical fungicides or solvents containing biocontrol strains would enhance their sterilizing effect. Since D-aas are non-toxic and stable, they are good candidates for the inhibition or destruction of plant pathogenic bacteria. However, whether D-Leu can effectively control harmful bacteria in the field requires further study. |
