Quorum Sensing Regulation Of Biocontrol Activity In Rhizobacterium Serratia Plymuthica HRO-C48

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). In this study, first of all, the antifungal activity of HRO-C48 against a variety of phytopathogenic fungi was examined in vitro. In greenhouse experiments, using mutational analysis by miniTn5 mutagenesis, and cucumber-Pythium aphanidermatum, tomato or bean-Botrytis cinerea as model systems, we explored the global quorum sensing regulation of biocontrol activity of HRO-C48 in vivo, in particularly focused on the rhizospheric colonization, the direct suppression of P. aphanidermatum in cucumber and the induced systemic resistance to B. cinerea. The data suggested that QS not only controlled the suppression of phytopathogenic fungi in vitro and in vivo, but also affected the interactions between HRO-C48 and the host plants, upregulated the ability of rhizosphere colonization and induced systemic resistance by HRO-C48. The major results achieved in detail are as follows:1. Using miniTn5 mutagenesis, 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 reporter. 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 suppressed a broad-spectrum of phytopathogenic fungi and formed different sizes of inhibition zone in dual culture with 14 fungi, such as Cryphonectria parasitica, Rhizoctonia cerealis and Valsa mali. More importantly, suprression of the phytopathogenic fungi, such as Valsa sordida and Botrytis cinerea by strain HRO-C48 in vitro is under the control of QS system as observed by mutational analysis.3. To determine the colonizations of HRO-C48, and its derivatives AHL-4 and AHL-4/splIR in the rhizosphere of both tomato and bean, the spread plate method was used on selective LA supplemented with rifampicin. Strain HRO-C48 successfully colonized rhizosphere of tomato and bean, and kept a stable population at concentration of 2×106 cfu/g at 4 weeks after soaking seeds and pouring root with biocontrol stains suspension. However, the ability of rhizosphere colonization for the mutant AHL-4 was decreased compared with HRO-C48, and restored to the level of the wild type when treated with the complementary AHL-4/splIR. It implied that QS controlled the colonization ability in rhizosphere of HRO-C48.4. 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 Pythium aphanidermatum in cucumber by HRO-C48. The biocontrol efficiency of HRO-C48, AHL-4 and AHL-4/splIR against cucumber damping-off were 52.38%, 28.57 and 50% respectively, compared with the tip water as control.5. The Botrytis cinerea-tomato or bean were used as model systems for further study of the potential of HRO-C48 to induce ISR. Strain HRO-C48 significantly reduced disease development in both two systems, but the mutant AHL-4 was deficient in the ability of ISR. However, the complementary strain AHL-4/splIR almost restored the induced resistance to B. cinerea to the level of wild type, which indicated that QS also positively regulate the induced systemic resistance of host plants.6. 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 activities of the PAL, POD and PPO were observed in leaves of tomato treated with B. cinerea, HRO-C48 and AHL-4, 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.