【作者】 楼妙苗；

【导师】 谢关林；

【作者基本信息】 浙江大学 ， 植物病理学， 2011， 博士

【摘要】 本研究以毛竹、雷竹以及桑树根围的土壤为主要对象,于2007-2010年间分别对其根围土壤中洋葱伯克氏菌群(Burkholderia cepacia complex,简称Bcc)的分布及其多样性进行了调查。采用洋葱、苜蓿和生菜致病模型对获得的Bcc菌作了致病毒力研究,探测不同植物根围来源以及不同基因型菌株间的毒力程度,为评估Bcc菌对人体的潜在致病性提供依据。鉴于杏果腐的病原细菌Burkholderia seminalis对人体存在的潜在威胁及目前尚无有效的防治方法,因此探索了壳聚糖对B. seminalis的抑菌效果及机制,以期找到安全有效防治Bcc菌引起的植物病害和人体病害的方法。主要取得了如下研究结果：(1)采用2种选择性培养基(PCAT和TB-T)对毛竹、雷竹和桑树根围土壤中的Bcc菌进行了调查,共分离到1127株Bcc疑似菌。通过对这些细菌进行recA基因的特异性扩增,共确定了666株Bcc菌,其中287株来源于毛竹根围土壤,222株来源于雷竹根围土壤,157来源于桑树根围土壤。采用限制性内切酶HaeⅢ以及MnlⅠ对来源于毛竹、雷竹和桑树根围的666株Bcc菌的recA基因片段进行了限制性酶切分析(RFLP)。结果表明,三种植物根围土壤来源的Bcc中,共发现了11种不同的HaeⅢ-RFLP图谱(D, E, G, H, J, T, M, K1,L1,L2和L3),其中7种图谱与文献报道的一致,4种(K1,L1,L2和L3)为本研究新发现的图谱。表明在这些环境中,Bcc菌群存在明显的遗传多态性。(2)采用recA-HaeⅢ限制性酶切分析、基因型特异性引物扩增,recA基因的序列分析和MLST技术对666株来源于毛竹、雷竹和桑树根围土壤来源的Bcc菌进行了种群分析。在毛竹根围土壤中存在B. stabilis, B. anthina, B. pyrrocinia和B. arboris 4个种,其中以B. pyrrocinia的菌株数量最多,占毛竹来源Bcc菌总数的67.2%；雷竹根围土壤中存在B. cepacia, B. anthina, B. pyrrocinia和B.arboris 4个种和Bcc group H,其中以Bcc group H和B. anthina菌株占多数,分别占雷竹来源Bcc菌总数的48.2%和38.3%；桑树根围来源的Bcc菌中,存在B. cepacia、B. cenocepaciaⅢA、B. cenocepaciaⅢB、B. stabilis、B. anthina、B. pyrrocinia、B. arboris和B. diffusa8个种,此外也有部分Bcc group H菌株,其中以B. cepacia为优势种,占桑树来源总Bcc菌数的61.1%。这是国内外首次报道B. pyrrocinia, Bcc group H和B. cepacia分别为毛竹、雷竹和桑树根围土壤中的优势种。采用多位点序列分型(MLST)技术对18株Bcc菌的代表菌株进行了序列分型研究,发现了17种不同的序列型(ST型),并且均为本研究新提交,说明环境来源的Bcc菌具有丰富的遗传多样性。(3)对分离自毛竹、雷竹和桑树根围的Bcc菌进行了洋葱、苜蓿和生菜的致病性研究。对洋葱鳞茎的致病力以B. pyrrocinia最强,9株未鉴定菌株最弱。对苜蓿幼苗以B. arboris的毒力最强而B. anthina最弱。对生菜的致病力以Bccgroup H和B. pyrrocinia最强,B. anthina几乎无致病毒力。总体而言,来源于根围土壤环境中Bcc菌对生菜的致病毒力较低,尤其是B. anthina对三种测试植株都不致病,表明环境来源的菌株对人体的风险较小。(4)本研究就三种壳聚糖对杏果腐病原菌B. seminalis的抑制效果进行了研究。此外,壳聚糖对B. seminalis的抑菌机理则通过细胞膜的完整性、细胞外膜的通透性、透射电镜观察以及对生物膜的抑制作用等实验进行探索。结果发现,2 mg/mL的酸溶性壳聚糖能有效抑制B. seminalis的生长；壳聚糖通过破坏细胞外膜,溶解细胞壁,造成细胞渗透压失常以及在细胞外形成壳聚糖层等多种方式抑制B. seminalis的生长；壳聚糖对B. seminalis的生物膜也有一定的抑制效果。实验结果表明,壳聚糖在保护果蔬不受B. seminalis的污染方面有很大的应用潜力。更多还原

【Abstract】 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 HaeⅢand MnlⅠ. The results showed 11 different HaeⅢ-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-HaeⅢRFLP 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. cenocepaciaⅢA, B. cenocepaciaⅢB, 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.更多还原