不同来源的洋葱伯克氏菌基因型对洋葱的致病性分析

通过对农田环境和医院的洋葱伯克氏菌基因型在洋葱上的致病性研究,结果表明,来源于根围的基因型Ⅲ和基因型Ⅰ的部分菌株,以及来源于医院的基因型Ⅰ和Ⅲ菌株均能使洋葱鳞茎腐烂,表现出与洋葱致病菌LMG1222相似的致病症状,但基因型Ⅴ和Ⅸ没有使洋葱发病。农田和医院环境中均存在对洋葱致病的洋葱伯克氏菌,而且各基因型对洋葱的致病性有差异。     

Burkholderia mallei infection in a horse imported from Brazil

A horse imported from Brazil developed a respiratory illness 2 weeks after arrival in Germany. After an initial but inefficient treatment glanders was diagnosed based on serological and molecular biological findings. The present case highlights the potential risk of an importation of glanders in free areas. The fact that veterinarians in countries where glanders has been eradicated for decades are not familiar with the clinical symptoms of the disease, can favour the entry of the disease. In order to prevent the spread of glanders, the sanctions of the veterinary authorities in such cases of the infection are of utmost importance.     

洋葱伯克霍尔德氏菌Lu10-1产生抗菌活性物质的发酵培养基和发酵条件优化

洋葱伯克霍尔德氏菌(Burkholderia cepacia)Lu10-1是从桑叶中分离得到的一株具有抗菌及促进植物生长等多种生物学功能的内生细菌。利用基于统计学的响应面法(response surface methodology,RSM)对影响该菌产生抗细菌活性物质的发酵培养基组成和发酵培养条件进行了优化。部分重复因子试验表明,酵母浸粉和氯化钠是培养基组分中的主要影响因子,其中酵母浸粉为正效应,氯化钠为负影响;结合最陡爬坡路径逼近最大响应区域和中心组合设计及响应面分析,确定了培养基中主要配方的最佳质量浓度为蔗糖17.0 g/L、酵母浸粉5.855 g/L、氯化钠4.519 g/L、磷酸二氢钾0.2 g/L。通过PB(plackeet-burman)试验发现接种量和发酵温度是该菌株产生抗菌活性物质发酵条件中的主要影响因子,经中心组合设计法优化的最佳发酵条件为:接种量0.027 7 mL/mL,摇瓶装液量100 mL,发酵温度30.29℃,培养基初始pH6.2,培养时间42 h。     

洋葱伯克霍尔德氏菌CAS19嗜铁素合成相关基因cepR的克隆及生物信息分析

本研究采用PCR方法克隆了洋葱伯克霍尔德氏菌CAS19嗜铁素合成相关基因cepR,并对cepR基因编码蛋白的结构和功能进行生物信息学分析和预测。同源性分析表明,CAS19的cepR核苷酸序列与Burkholderiacepacia LMG1222cepR基因的同源性为99％;cepR基因全长768bp,包含一个完整的231bp的开放阅读框架,编码239个氨基酸;该基因编码蛋白分子量为26．59kD,理论的等电点为5．55,含有一个LuxR型HTH结构域,在氨基酸残基的不同区域分布有酪蛋白激酶Ⅱ磷酸化位点、蛋白激酶C磷酸化位点、酪氨酸激酶磷酸化位点、N-肉豆蔻酰化位点和N-糖基化位点,还有一个明显的跨膜结构。本研究结果将为洋葱伯克霍尔德氏菌嗜铁素合成相关研究提供调控基因信息和参考依据,并为其进一步开发和利用奠定基础。     

Association between Burkholderia species and arbuscular mycorrhizal fungus spores in soil

Burkholderia pseudomallei, the bacterial cause of the potentially fatal infection known as melioidosis, has a facultative intracellular lifestyle. The intracellular presence of B. pseudomallei in various eukaryotes including arbuscular mycorrhizal fungus (AMF) spores can be demonstrated in vitro. AMF spores were isolated from soils in a melioidosis-endemic area. B. pseudomallei and other Burkholderia spp. DNA was detected in these AMF spore samples, confirming an AMF spore-Burkholderia spp. association in soils which did not yield Burkholderia spp. by culture. This association may explain the environmental persistence, difficulty of recovery and dispersal of Burkholderia spp. in specific environments.     

采前喷施洋葱伯克霍尔德菌Burkholderia contaminans对草莓采后腐烂和品质的影响

为明确采前喷施洋葱伯克霍尔德菌Burkholderia contaminans B-1对草莓采后贮藏期的腐烂和品质的影响,采用洋葱伯克霍尔德菌菌悬液喷施草莓植株,检测草莓采后腐烂率及硬度、色泽、可滴定酸、抗坏血酸及失重率等指标,并统计草莓表面菌落生长情况。结果表明,采前喷施浓度为1×10~(10)CFU/m L的洋葱伯克霍尔德菌B-1菌悬液,可显著降低草莓采后腐烂的发生,而且喷施3次贮藏效果优于喷施1次。在16℃下贮藏7 d,喷施3次处理在接种灰葡萄孢菌和自然条件下的抑制率分别达到67.33%和58.45%。采前喷施洋葱伯克霍尔德菌菌悬液可有效提升草莓品质,延缓果实硬度、可溶性固形物、可滴定酸,降低Vc含量,一定程度上可保持果实亮度。洋葱伯克霍尔德菌可在果实表面定殖,且抑制果实表面病原菌的生长。菌悬液喷施3次,16℃贮藏7 d后,果实表面拮抗菌数量达到贮藏前的4.79×10~4倍,同时,病原菌在果实表面的菌落数也大大减少。洋葱伯克霍尔德菌可引起灰葡萄孢菌菌丝畸形,内含物外渗。此外,洋葱伯克霍尔德菌对草莓植株白粉病有抑制作用,并促进植株生长,株高、单株鲜重和单株叶片分别比对照增加19.39%、28.13%和36.04%。研究表明该菌株在草莓生产和贮藏领域具有较大潜力。     

Cloning,Prokaryotic Expression and Bioinformatics Analysis of BPSS0180 Gene of Burkholderia pseudomallei

This study was aimed to clone and express the BPSS0180 gene of Burkholderia pseudomallei (B. pseudomallea), and perform bioinformatics analysis of its protein. A pair of primers was designed according to the BPSS0180 gene sequence information of B. pseudomallea K96243 strain in GenBank. BPSS0180 gene fragment was obtained by PCR amplification of B. pseudomallea hn-1 strain. The BPSS0180 gene fragment was ligated into the pET-28a(+) vector to construct the pET-28a(+)-BPSS0180 recombinant plasmid. The recombinant plasmid pET-28a(+)-BPSS0180 was transformed into E.coli DH5α competent cells, and the plasmids were identified by restriction enzyme digestion. Then, the recombinant plasmid pET-28a(+)-BPSS0180 was transformed into E.coli BL21 (DE3) competent cells. The expression was induced by IPTG. The expressed product was analyzed by SDS-PAGE and Western blotting. Bioinformatics analysis of BPSS0180 gene sequence was carried out using DNAMAN, ProtParam, SOPMA and Protscale. The results showed that the length of BPSS0180 gene was 1 146 bp; The expressed His-BPSS0180 fusion protein was about 45 ku, and was predominantly in the form of inclusion bodies; The molecular weight of the BPSS0180 protein was 40.6 ku (C₁₇₇₉H₂₈₀₉N₅₄₅O₅₃₆S₇); The extinction coefficient was 40 575; The hydrophobic index was 85.43; The instability coefficient was 46.52,which belonged to unstable protein;The theoretical isoelectric point (pI) was 5.54 and was acidic protein; The total average hydrophobicity (GRAVY) was -0.261,as hydrophilic protein; The secondary structure of the protein were mainly α-helix (58.79%) and random curl (32.02%), and its half-life of reticulocytes in mammals was predicted to be 30 h. This study provided a theoretical basis for further exploring the fuction of BPSS0180 gene of B. pseudomallei.     

双重PCR方法检测检疫性细菌洋葱腐烂病菌和菊基腐病菌

为建立同步检测检疫性细菌洋葱腐烂病菌和菊基腐病菌的双重PCR方法,根据GenBank上公布的洋葱腐烂病菌ITS序列设计1对特异性引物B3/B6,将设计的引物与已发表的检测菊基腐病菌特异性引物ADE1/ADE2结合,经过条件优化,建立了双重PCR反应体系,并进行特异性和灵敏度验证。应用双重PCR体系能从洋葱腐烂病菌和菊基腐病菌基因组DNA以及人工带菌样品中扩增到特异性条带。结果表明,建立的双重PCR检测方法能同时检测出洋葱腐烂病菌和菊基腐病菌,可应用于口岸进口郁金香种球2种检疫性细菌的检测。     

New method to detect oxolinic acid-resistant <Emphasis Type="Italic">Burkholderia glumae</Emphasis> infesting rice seeds using a mismatch amplification mutation assay polymerase chain reaction

Bacterial seedling rot and grain rot of rice caused by Burkholderia glumae are seed-borne diseases, traditionally controlled with oxolinic acid (OA). Ser83Arg and Ser83Ile substitutions in GyrA protein are commonly responsible for moderate and high resistance to OA, respectively, in field isolates of B. glumae. To detect OA-resistant B. glumae infesting rice seeds, a mismatch amplification mutation assay polymerase chain reaction protocol was developed using DNA from bacteria incubated in modified S-PG medium and primers based on the amino acid substitutions at position 83 in GyrA.     

Analysis of sources of oxolinic acid-resistant field strains of <Emphasis Type="Italic">Burkholderia glumae</Emphasis> based on rep-PCR analysis and nucleotide sequences of <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">rpoD</Emphasis>

Repetitive sequence-based polymerase chain reaction (rep-PCR) analysis using BOX and ERIC as primers showed a highly divergent phylogeny among field strains of Burkholderia glumae. To elucidate the sources of oxolinic acid (OA) resistance in field strains of B. glumae isolated from rice seedlings cultivated in Mie, Toyama, and Iwate prefectures, Japan, the amino acid at position 83 of GyrA (GyrA83), which is involved in OA resistance, and the DNA patterns from the rep-PCR and the partial nucleotide sequences of gyrB and rpoD from various strains were analyzed. The ten Mie strains, in which GyrA83 was isoleucine (Ile), were divided into two groups based on the band patterns in rep-PCR analysis, although the nucleotide sequences of gyrB and rpoD were identical among the strains. Based on the band patterns in the rep-PCR analysis and the gyrB and rpoD sequences, two highly OA-resistant Toyama strains, Pg-13 and Pg-14, for which GyrA83 was serine (Ser) and Ile, respectively, were in the same lineage. This suggests that the bacteria might acquire OA resistance faster than phylogenic diversity as determined with the repetitive sequences BOX and ERIC and with gyrB and rpoD. Furthermore, three Iwate strains (H95, H101, and H104), isolated from seedlings of different cultivars grown in different years and having Ile at GyrA83, are probably in the same lineage, suggesting that OA-resistant bacteria might be transferred among different cultivars.