The Role Of Chitinases And Quorum Sensing Signals In Biocontrol By Serratia Plymuthica

Serratia plymuthica HRO-C48 was isolated from the rhizosphere of oilseed rape in Germany and displayed biocontrol potential to suppress several plant diseases due to production of chitinases, protease and pyrrolnitrin, as well as promoting plant growth by production of indole acic acid (IAA). Using mutational analysis by miniTn5 mutagenesis, we explored the role of chitinase and quorum sensing signals in biocontrol activity of HRO-C48 in vitro and in vivo; The second luxIR homologous genes sprRI of strain HRO-C48 were cloned, followed by heterologous expression in E. coli JM109 carrying plasmid pMD19-T-sprRI. The major results achieved in detail are as follows:1. Using miniTn5 mutagenesis, chitinase-deficient mutant (chit) was screened on selective SM medium with 0.2% colloidal chitin as the sole carbon sources by testing their ability to produce chitinases. AHL-deficient mutant (AHL-4) was screened on selective LA supplemented with rifampicin and kanamycin plates and tested for their ability to produce AHLs in an on-plate assay using Chromobacterium violaceum CV026 as the bioreporter. The genes splI and splR, which are analogues of luxI and luxR genes from other gram-negative bacteria, were cloned and sequenced (The GenBank accession number is AY841161). AHL-4 complementation strain (AHL-4/splIR) carrying plasmid pUCP26-splIR was constructed. All derivative strains were verified by restriction analysis, PCR and gene mapping.2. Confrontation bioassay of antifungal activity on PDA plates showed that strain HRO-C48 and its derivatives suppressed several common phytopathogens, such as Botrytis cinerea and Pythium aphanidermatum. The antifungal activity of AHL-4 was less than that of HRO-C48, but for AHL-4/splIR and chit, the antifungal activities were the same as the HRO-C48's. The data implied that the suprression of the phytopathogenic fungi by strain HRO-C48 in vitro is under the control of QS signals and that the antibiotics are important in the antagonism of HRO-C48.3. The B. cinerea-tomato or bean was used as model systems for further study of the ISR by HRO-C48. Strain HRO-C48 significantly reduced disease development in both two systems, but the mutant AHL-4 was deficient in the ability of ISR. It indicated that QS also positively regulate the induced systemic resistance of host plants. However, the ability of ISR was distinguishing between HRO-C48 and the mutant chit only in the system of the B. cinerea-tomato.4. After the same treatment as described above, cucumber seedlings were inoculated with P. aphanidermatum and the disease incidence of damping-off was examined at three or four days after inoculation of pathogen. Results showed that QS affected the suppression of P. aphanidermatum in cucumber by HRO-C48. The biocontrol efficiency of HRO-C48 and AHL-4 against cucumber damping-off were 50.24% and 27.64% respectively, compared with the tip water as control. However, the biocontrol efficiency of the mutant chit was 37.30%, which was not the same as that of HRO-C48. It implied that the activity of ISR by chitinases was included in the control of the damping-off for the cucumber seedlings.5. The content of phenolic compounds and the activities of plant defence related enzymes, such as PAL, POD and PPO were examined after treatment with biocontrol strain suspension and B. cinerea inoculation, the increases of content of phenolic compounds and activities of the PAL, POD and PPO were observed in leaves of tomato treated with B. cinerea, HRO-C48, AHL-4, AHL-4/splIR and chit, compared to the control tomato leaves treated with tip water. It suggested that the ISR by HRO-C48 was related to activate the defence reaction of the host plant. 6. The second luxIR homologous genes sprRI of strain S. plymuthica HRO-C48 based on the conserved primers designed by consensus sequences of luxIR in related genera with the genomic DNA of HRO-C48 as template, followed by heterologous expression in E. coli JM109 carrying plasmid pMD19-T-sprRI. Comparison of the reported homologue genes and phylogenetic analysis using bioinformatics showed higher similarites to the other luxIR homologous genes in related genera.