Diversity And Virulence Of Burkholderia Cepacia Complex Around The Plant Rhizosphere, Antibacterial Mechaism Of Chitosan Against B. Seminalis

An survey of the distribution and diversity of Burkholderia cepacia complex (Bcc) around the rizhosphere soil of moso bamboo(Phyllostachys edulis), Phyllostachys praecox f. prevernalis (PPFP) and mulberry (Morus alba L.) were conducted during 2007-2010; Bcc isolates were identified and the predominant Bcc species were determined in the rizhosphere environment. The pathogenicity of Bcc was evaluated using different plant infection models, including onion, alfalfa and lettuce detached midrib in order to provide key information in risk analysis of Bcc. Apricot rot pathogen B. seminalis was also potentially pathogenic to human being, since no pesticide is available to inhibit the growth of Bcc particularly B. seminalis, the antibacterial activity and mechanism of action of chitosan solutions against apricot fruit rot pathogen B. seminalis were examined at present study. The main results were as follows:(1) Two selective medium PC AT and TB-T were used for isolation of Bcc from rizhosphere soil of moso bamboo, PPFP and mulberry. Among the 1127 isolates,666 isolates were identified as Bcc, and 287,222 and 157 isolates were obtained from rizhosphere soil of moso bamboo, PPFP and mulberry, respectively.The genetic variability of 666 strains from rizhosphere soil of moso bamboo, PPFP and mulberry were anlysised by restriction fragment length polymorphism assays (RFLP) with enzyme Haeâ…¢and Mnlâ… . The results showed 11 different Haeâ…¢-RFLP patterns (D, E, G, H, J, T, M, K1, L1, L2, L3) among the 666 Bcc isolates, seven patterns were consistent with previous reports while four (K1, L1, L2 and L3) were newly found in our study, indicating considerable variability among the Bcc isolates.(2) The species identification of the 666 Bcc isolates from the rhizosphere soil of moso bamboo, PPFP and mulberry were performed by a combination of recA-Haeâ…¢RFLP assays, species-specific PCR tests, recA gene sequence analysis and multilocus sequence typing scheme (MLST). Four species including B. stabilis, B. anthina, B. pyrrocinia and B. arboris were recovered from the moso bamboo rizhosphere soil with predominace of B. pyrrocinia accounting for 67.2% of the total. Four species including B. cepacia, B. anthina, B. pyrrocinia and B. arboris as well as Bcc group H were recovered from the PPFP rizhosphere soil. The predominace species of Bcc isolates from PPFP were Bcc group H and B. anthina accounting for 48.2% and 38.3% of the total, respectively. Bcc group H and eight species including B. cepacia, B. cenocepaciaâ…¢A, B. cenocepaciaâ…¢B, B. stabilis, B. anthina, B. pyrrocinia, B. arboris and B. diffusa were found among the isolates from mulberry rizhosphere soil, and B. cepacia was predominant with 61.1% of the total. It was the first report about B. pyrrocinia, Bcc group H and B. cepacia as the predominant species of the rizhosphere soils of moso bamboo, PPFP and mulberry, respectively.The Multilocus Sequence Typing (MLST) scheme was performed within 18 Bcc representatives; all were successfully sequenced at all seven loci, resulting in a total of 17 STs. All the 17 STs were newly submitted indicating considerable strain diversity of Bcc in soil environment.(3) The pathogenicity to onion and virulence to alfalfa and lettuce of Bcc from rizhosphere of moso bamboo, PPFP and mulberry was examined. B. pyrrocinia showed highest pathogenicity to onion, B. arboris were most virulent to alfalfa while Bcc group H and B. cepacia were most virulent to lettuce. B. anthina were almost non-pathogenic or non-virulent to onion, alfalfa, and lettuce. In general, most isolates tested in our study showed none, weak or moderate virulence especially B. anthina which is almost non-pathogenic or non-virulent to all test plants, suggested that environmental strains may not be so dangerous to human beings.(4) The in vitro antibacterial activity and mechanism of action of three kinds of chitosan was examined in this study. Results showed that water-soluble chitosan displayed limited antibacterial activity while two kinds of acid-soluble chitosan solution at 2.0 mg/mL had strong antibacterial activity against B. seminalis. The antibacterial activity of acid-soluble chitosan may be due to the membrane disruption, cell lysis, abnormal osmotic pressure, and additional chitosan coating around the bacteria based on integrity of cell membranes test, out membrane permeability assays and transmission electron microscopy observation. In addition, biofilm biomass were markedly reduced after treating with two kinds of acid-soluble chitosan. Overall, the results clearly indicated that two kinds of acid-soluble chitosan had a potential to control the contamination of apricot fruits caused by B. seminalis.