抗生素2,4-二乙酰基间苯三酚作为荧光假单胞菌2P24菌株生防功能因子的实证分析

荧光假单胞杆菌2P24菌株分离自小麦全蚀病自然衰退土壤,它是酚类抗生素2,4-二乙酰基间苯三酚(2,4-DAPG)的高产菌,对多种土传病害具有较好的防治能力。利用同源重组构建2,4-DAPG合成基因的定位突变体,并对突变体进行基因互补,通过检测突变菌株和恢复突变菌株抗生素产量和生防效果确定2,4-DAPG在菌株2P24生防功能中的作用。实验中,定位突变体丧失产生抗生素和拮抗病原菌的能力,而恢复突变体的抗生素产量和拮抗能力均恢复至野生菌水平。在对番茄青枯病的防病试验中,2,4-DAPG突变体的防效低且下降快,而恢复突变体的生防能力与野生菌相当,且效果稳定。由此可确定2,4-DAPG是菌株2P24防治番茄青枯病的主要因子,在防效上起关键作用。     

烟草青枯雷尔氏菌噬菌体资源的发掘

由青枯雷尔氏菌(Ralstonia solanacearum)引起的烟草青枯病是烟草种植过程中一种重要的细菌性病害。为了开展利用噬菌体防控烟草青枯病的研究,本研究以烟田产区的7株烟草青枯菌为宿主,从烟田土壤中分离到了14个对烟草青枯菌具有侵染性的烈性噬菌体,说明烟田土壤中普遍存在烟草青枯菌噬菌体。选取噬菌体?PB2和?PB34,电镜观察确定其具有十二面体的头部和粗短的尾部,属于肌尾噬菌体科。噬菌体的宿主谱分析显示,筛选到的噬菌体对分别从烟草、西红柿和花生分离的青枯菌株系的侵染性有所差异。本研究分离发掘的14个对烟草青枯菌侵染的噬菌体,为利用噬菌体防控烟草青枯病的生防策略研究提供了噬菌体资源。     

Transport of bacterial inoculants through intact cores of two different soils as affected by water percolation and the presence of wheat plants

Abstract Water flow-innduced transport of Burkholderia cepacia strain P2 and Pseudomonas fluorescens strain R2f cells through intact cores of loamy sand and silt loam field soils was measured for two percolation regimes, 0.9 and 4.4 mm h⁻¹, applied daily during 1 hour. For each strain, transport was generally similar between the two water regimes. Translocation of B. cepacia , with 4.4 mm h⁻¹, did occur initially in both soils. In the loamy sand soil, no change in the bacterial distribution occurred during the experiment (51 days). In the silt loam, B. cepacia cell numbers in the lower soil layers were significantly reduced, to levels at or below the limit of detection. Transport of P. fluorescens in both soils also occurred initially and was comparable to that of B. cepacia . Later in the experiment, P. fluorescens was not detectable in the lower soil layers of the loamy sand cores, due to a large decrease in surviving cell numbers. In the silt loam, the inoculant cell distribution did not change with time. Pre-incubation of the inoculated cores before starting percolation reduced B. cepacia inoculant transport in the loamy sand soil measured after 5 days, but not that determined after 54 days. Delayed percolation in the silt loam soil affected bacterial transport only after 54 days. The presence of growing wheat plants overall enhanced bacterial translocation as compared to that in unplanted soil cores, but only with percolating water. Percolation water from silt loam cores appeared the day after the onset of percolation and often contained inoculant bacteria. With loamy sand, percolation water appeared only 5 days after the start of percolation, and no inoculant bacteria were found. The results presented aid in predicting the fate of genetically manipulated bacteria in a field experiment.     

Characterization of filamentous bacteriophage PE226 infecting Ralstonia solanacearum strains

Aims: The aim of this study was to isolate and characterize new bacteriophages that infect a wide range of plant pathogenic Ralstonia solanacearum strains. Methods and Results: Fifteen bacteriophages were isolated from pepper, tomato and tobacco plant rhizospheres infected with R. solanacearum. A host specificity analysis of the isolated phages using nine strains of R. solanacearum indicated great phage diversity in a single soil. Two phages, PE226 and TM227, showed clear plaques on all nine bacterial hosts tested and were virtually identical in morphology and genome. PE226, an Inovirus, is a long, flexible, filamentous phage carrying a circular (+) sense single‐strand DNA genome of 5475 nucleotides. DNA sequences of PE226 exhibited nine open reading frames (ORF) that were not highly similar to those of other phages infecting R. solanacearum. The genome organization of PE226 was partially similar to that of p12J of Ralstonia pickettii. One ORF of PE226 showed identity to the zot gene encoding zonula occludens toxin of Vibrio cholera. Orf7 of PE226 was also present in the genome of R. solanacearum strain SL341. However, SL341, a highly virulent strain in tomato, was still sensitive to phage PE226. Conclusions: A new, flexible, filamentous phage PE226 infected wide range of R. solanacearum strains and carried unique circular single‐strand DNA genome with an ORF encoding Zot‐like protein. Significance and Impact of the Study: PE226 may be a new type of temperate phage, based on its lytic nature on a wide range of hosts and the presence of a zot homologue in a host bacterial genome.     

云南省烟植地青枯菌RS-22的分离及其拮抗菌的筛选和鉴定

【背景】由青枯劳尔氏菌(Ralstonia solanacearum)引起的烟草青枯病是一种重要土传病害,在我国南方烟区普遍发生。生物防控是针对烟草青枯病的一种有效防治措施,但是相关的研究报道还较少。【目的】分离云南省烟植地的青枯病原菌,筛选其拮抗菌并对其抑菌效果进行鉴定。【方法】采用平板稀释法从云南感病烟草中分离获得青枯菌,采用平板对峙法筛选青枯菌拮抗菌,筛选得到的拮抗菌通过16SrRNA基因测序比对确定菌种类型,并在实验室和大田鉴定其对青枯病的防治效果。【结果】从感病烟草茎中分离出一株强致病性青枯菌小种RS-22,该菌能侵染烟草和番茄并最终使植物死亡;筛选出12株RS-22拮抗菌,其中拮抗作用最强的是Y4;Y4被鉴定为一株解淀粉芽孢杆菌(Bacillus amyloliquefaciens),其菌体和分泌物都能抑制RS-22生长;Y4根部灌根处理能显著提高烟草和番茄对青枯菌RS-22的抗性,Y4处理能使感病烟草部分恢复正常,在云南文山州烟草种植大田施加Y4菌剂和菌剂有机肥混合物也能显著降低烟草青枯病的感病率。【结论】青枯菌RS-22具有广谱的致病性,筛选的拮抗菌Y4能显著抑制青枯菌生长,而且对青枯菌侵染植物有很好的防治效果。研究结果为进一步研究烟草青枯病的生物防控提供了新的理论依据。     

Ethyl β-d-glucoside: a novel chemoattractant of Ralstonia solanacearum isolated from tomato root exudates by a bioassay-guided fractionation

ABSTRACT

A chemoattractant of Ralstonia solanacearum isolated from the activated charcoal-adsorbed fraction of tomato root exudates was identified as ethyl β-d-glucopyranoside by instrumental analyses and comparison with synthetic preparations. Ethyl β-D-glucopyranoside showed unambiguous activity at above 1 µmol/disc. Its stereoisomers and D-glucose were inactive.     

油松菌根促生细菌——荧光假单胞菌的分离与鉴定

为了探讨菌根促生细菌荧光假单胞菌(Pseudomonas fluorescens)与菌根真菌的互作关系,本实验从油松菌根上分离得到36株在紫外灯下产荧光的细菌菌株,以荧光假单胞菌9702作为标准菌株,对分离菌株进行显微观察、生物学鉴定和16S rDNA序列分析,结果确定HDY-8、HDY-9、HDY-20、HDY-35共 4株菌株为荧光假单胞菌,并分别命名为P.fluorescens HDY-8、P.fluorescens HDY-9、P.fluorescens HDY-20、P.fluorescens HDY-35.用这4株细菌菌株分别与外生菌根真菌(ECMF)粘盖牛肝菌(Suillus bovinus)、褐环粘盖牛肝菌(Suillus luteus)和褐黄牛肝菌(Boletus luridus)进行纯培养互作研究.结果表明,只有P.fluorescens HDY-20对3种外生菌根真菌有不同程度的促生作用,并对S.luteus促进效果最好,S.bovinus次之,B.luridus最差;P.fluorescens HDY-20促进S.bovinus、S.luteus和B.luridus菌丝生长的最佳浓度分别为2.4×109 cfu/mL、0.8×109～2.4×109 cfu/mL和0.8×109 cfu/mL,与对照相比S.bovinus和S.luteus的生物量达到极显著差异(P<0.01),B.luridus的达到显著差异(P<0.05),且分别比对照增加6.5%、9.1%和4.3%.     

Optimization of the hide powder azure assay for quantitating the protease of Pseudomonas fluorescens

The extracellular protease of Pseudomonas fluorescens NC 3 was optimally active at 40°C in a reaction mixture containing: 50 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer (pH 6.6), 0.5 mM CaCl₂, and 25 mg hide powder azure in 5 ml total volume. Divalent cation chelators, i.e., EDTA, o-phenanthroline, citrate or phosphate, inhibited the enzyme. Protease production by P. fluorescens NC 3 was initiated during late-logarithmic-growth phase in a sodium caseinate medium and reached its maximum at the onset of the stationary phase.     

Characterization of initial events in bacterial surface colonization by two Pseudomonas species using image analysis

The processes leading to bacterial colonization on solidwater interfaces are adsorption, desorption, growth, and erosion. These processes have been measured individually in situ in a flowing system in real time using image analysis. Four different substrata (copper, silicon, 316 stainless-steel and glass) and 2 different bacterial species (Pseudomonas aeruginosa and Pseudomonas fluorescens) were used in the experiments. The flow was laminar (Re = 1.4) and the shear stress was kept constant during all experiments at 0.75 N m(-2). The surface roughness varied among the substrata from 0.002 mum (for silicon) to 0.015 mum (for copper). Surface free energies varied from 25.1 dynes cm(-1) for silicon to 31.2 dynes cm(-1) for copper. Cell curface hydrophobicity, reported as hydrocarbon partitioning values, ranged from 0.67 for Ps. fluorescens to 0.97 for Ps. aeruginosa.The adsorption rate coefficient varried by as much as a factor of 10 among the combinations of bacterial strain and substratum material, and was positively correlated with surface free energy, the surface roughness of the substratum, and the hydrophobicity of the cells. The probability of desorption decreased with increasing surface free energy and surface roughness of the substratum. Cell growth was inhibited on copper, but replication of cells overlying an initial cell layer was observed with increased exposure time to the cell-containing bulk water. A mathematical model describing cell accumulation on a substratum is presented.     

Endophytic colonization of olive roots by the biocontrol strain Pseudomonas fluorescens PICF7

Confocal microscopy combined with three-dimensional olive root tissue sectioning was used to provide evidence of the endophytic behaviour of Pseudomonas fluorescens PICF7, an effective biocontrol strain against Verticillium wilt of olive. Two derivatives of the green fluorescent protein (GFP), the enhanced green and the red fluorescent proteins, have been used to visualize simultaneously two differently fluorescently tagged populations of P. fluorescens PICF7 within olive root tissues at the single cell level. The time-course of colonization events of olive roots cv. Arbequina by strain PICF7 and the localization of tagged bacteria within olive root tissues are described. First, bacteria rapidly colonized root surfaces and were predominantly found in the differentiation zone. Thereafter, microscopy observations showed that PICF7-tagged populations eventually disappeared from the root surface, and increasingly colonized inner root tissues. Localized and limited endophytic colonization by the introduced bacteria was observed over time. Fluorescent-tagged bacteria were always visualized in the intercellular spaces of the cortex region, and no colonization of the root xylem vessels was detected at any time. To the best of our knowledge, this is the first time this approach has been used to demonstrate endophytism of a biocontrol Pseudomonas spp. strain in a woody host such as olive using a nongnotobiotic system.     

鳜源致病性荧光假单菌的分离与鉴定

为确定导致鳜（Siniperca chuatsi）细菌性感染死亡的病原,从患病濒死的鳜肝中分离出一株优势菌B01,经人工回感实验确定其分离菌的致病性,并利用VITEK-2全自动微生物鉴定仪及16S rRNA序列分析对纯培养细菌进行鉴定。此外,对分离的菌株进行药敏实验。研究结果表明,菌株B01对鳜鱼具有致病性。经VITEK-2全自动微生物鉴定仪鉴定菌株B01为荧光假单胞菌（Pseudomonas fluorescens）（表1）,并在紫外灯可以产生荧光（图1）。进一步的16S rRNA基因序列和系统发育树分析表明,该菌与荧光假单胞菌同源性达到了97%以上,并和（图2）。药物敏感性实验结果显示,该分离株对恩诺沙星、诺氟沙星、链霉素和四环素药物等6种药物高度敏感（表2）。