菠菜内生固氮菌的分离及其功能特性研究

内生固氮菌具有促生、病原菌的防治及生物固氮等作用,并且占据着植物组织内有利于营养供应和微环境适宜的生态位,是重要的微生物菌种资源。从菠菜根内分离筛选优势内生固氮菌7株,其中假单胞菌属(Pseudomonas)5株,红球菌属(Rhodococcus)和黄杆菌属(Flavobacterium)各1株。菌株Pseudomonas sp.BCE6和Pseudomonas sp.BC-E8固氮酶活性较高,分别为(13.19±0.32)和(12.11±0.96)C2H4nmol/(mg protein·h),与圆褐固氮菌(ACCC11103)的固氮酶活性具有极显著性差异(P0.01);菌株Pseudomonas sp.BC-E7具有固氮酶活性[(8.42±0.03)C2H4nmol/(mg protein·h)]、产IAA能力[(59.58±4.15)μg/m L]、ACC脱氨酶活性[(5.067±0.376)μmol/(mg protein·h)],拮抗立枯丝核菌ACCC36124(Rhizoctonia solani)、麦类赤霉病ACCC36272(Gibberella zeae)等4种常见土传病害。菠菜内生固氮菌Pseudomonas sp.BC-E7是一株多功能内生菌,具有较好的生产应用前景。     

低温热处理对超高温灭菌乳中假单胞菌脂肪酶的活性影响

通过分析不同因素对假单胞菌脂肪酶活性的影响以及假单胞菌脂肪酶活性与超高温灭菌(UHT)乳脂肪上浮的关系,研究了控制UHT乳中假单胞菌脂肪酶活性和延缓脂肪上浮现象的措施。结果表明pH值对假单胞菌脂肪酶的活性有显著性影响(P<0.01),50、55、60℃(20min)的低温热处理能显著降低假单胞菌脂肪酶的活性(P<0.01)。贮藏试验表明低温热处理对控制UHT乳在贮存过程中的假单胞菌脂肪酶活性水平(P<0.01)和脂肪上浮现象(P<0.05)有明显的作用,而55℃和15min的低温热处理对缓解UHT乳在贮藏过程中的脂肪上浮现象最为有效。     

Suppression of root-knot disease by Pseudomonas fluorescens CHA0 in tomato: importance of bacterial secondary metabolite, 2,4-diacetylpholoroglucinol

The antimicrobial metabolites 2,4-diacetylphloroglucinol (2,4-DAPG) and pyoluteorin contribute to the ability of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soil-borne pathogens. P. fluorescens strain CHA0 and its derivatives CHA89 (antibiotics-deficient) and CHA0/pME3424 (antibiotics overproducing) were investigated as potential biocontrol agents against Meloidogyne javanica the root-knot nematode. Exposure of root-knot nematode to culture filtrates of P. fluorescens under in vitro conditions significantly reduced egg hatch and caused substantial mortality of M. javanica juveniles. Nutrient broth yeast extract (NBY) medium amended with 2% (w/v) glucose or 1 mM EDTA markedly repressed hatch inhibition activity of the strain CHA0 but not that of CHA0/pME3424 or CHA89. On the other hand, NBY medium amended with glucose significantly enhanced nematicidal activity of the strain CHA0/pME3424. Neither glucose nor EDTA had an influence on the nematicidal activity of the strains CHA0 and CHA89. Under in vitro conditions, antibiotic overproducing strain CHA0/pME3424 and CHA0 expressed phl‘-’lacZ reporter gene but strain CHA89 did not. Expression of the reporter gene reflects actual production of DAPG. In general, CHA0/pME3424 expressed reporter gene to a greater extent compared to its wild type counterpart CHA0. Regardless of the bacterial strains, reporter gene expression was markedly enhanced when NBY medium was amended with glucose but EDTA had no such effect. A positive correlation between the degree of juvenile mortality and extent of phl‘-’lacZ reporter gene expression was also observed in vitro. Strain CHA0 produced zones of 4-6 mm on MM medium containing gelatin while strain CHA0/pME3424 and CHA89 did not. When MM medium containing gelatin was amended with 2% glucose of 1 mM EDTA size of haloes produced by the strain CHA0 reduced to 2 mm. Under glasshouse conditions aqueous cell suspension of the strains CHA0 or CHA0/pME3424 at various inoculum levels (10⁷, 10⁸ or 10⁹ cfu ml⁻¹) significantly reduced root-knot development. CHA89 caused significant reduction in galling when applied at 10⁹ cfu ml⁻¹. To better understand the mechanism of nematode suppression, split root bioassay was performed. Split-root experiments, that guarantee a spatial separation of inducing agent and a challenging pathogen, showed that soil treatment of one half of the root system with cell suspension of CHA0 or CHA0/pME3424 resulted in a significant systemic induced resistance leading to reduction of M. javanica infection of tomato roots in the non-baterized nematode treated half. The results clearly suggest that the antibiotic 2,4-DAPG from P. fluorescens CHA0 act as the inducing agents of systemic resistance in tomato roots. Populations of CHA0 and its derivatives declined progressively by 10-fold between first and fourth harvests (0-21 days after inoculation). However, bacterial populations increased at final harvest (28 days after application).     

Colonization of wheat seedling (Triticum aestivum) roots by Pseudomonas fluorescens and Bacillus subtilis

Summary Colonization patterns of Pseudomonas fluorescens and Bacillus subtilis on roots of wheat seedlings growing on water agar were studied qualitatively by replica printing and quantitatively by the plate count method. The results indicated a stronger colonization potential for P. fluorescens (up to 10⁷ cfu/cm root) than for B. subtilis (up to 10⁵ cfu/cm root). Although the numbers of both species were lower when inoculated together, the observed colonization patterns on the roots were comparable to those found with single inoculations. For none of these bacteria was active migration along the root surface in any direction observed, indicating that distal positions are reached mainly by a passive displacement on the root tip and elongating cells. Ecological implications of the observed phenomena are discussed.     

Response of spring wheat (Triticum aestivum) to interactions between Pseudomonas species and Glomus clarum NT4

The effects of interactions between pseudomonads (Pseudomonas cepacia strains R55 and R85, P. aeruginosa strain R80, P. fluorescens strain R92, and P. putida strain R104) and the arbuscular mycorrhizal fungus Glomus clarum (Nicol. and Schenck) isolate NT4, on spring wheat (Triticum aestivum L. cv. Laura), grown under gnotobiotic and nonsterile conditions, were investigated. Although plant growth responses varied, positive responses to pseudomonad inoculants generally were obtained under gnotobiotic conditions. Shoot dry weight enhancement ranged from 16 to 48%, whereas root enhancement ranged from 82 to 137%. Shoot growth in nonsterile soil, however, was unaffected by pseudomonad inoculants, or reduced by as much as 24%. Shoot growth was unaffected or depressed by G. clarum NT4 whereas early root growth was enhanced by 38%. Significant interactions between the pseudomonad inoculants and G. clarum NT4 were detected. Typically, dual inoculation influenced the magnitude of response associated with any organism applied alone. The effect of these pseudomonads on G. clarum NT4 spore germination was investigated. Germination was inhibited when spores were incubated either on membranes placed directly on bacterial lawns of strains R85 and R104 (i.e., direct assay), or on agarose blocks separated from the bacteria by membranes (i.e., diffusion assay). When the agarose blocks were physically separated from the pseudomonad (i.e., volatile assay), there was no evidence of inhibition, suggesting that a nonvolatile, diffusible substance(s) produced by both strains R85 and R104 may inhibit G. clarum NT4 spore germination. Received: 11 December 1995     

Fate, tree growth effect and potential impact on soil microbial communities of mycorrhizal and bacterial inoculation in a forest plantation

The knowledge of the survival of inoculated beneficial fungal and bacterial strains in the field and the effects of their release on the indigenous microbial communities has been of great interest since the practical use of selected natural or genetically modified microorganisms has been developing. The aim of this study was to monitor, 4 years after plantation into the field site, the effects of Douglas fir (Pseudotsuga menziesii) co-inoculation with the mycorrhiza helper bacterial strain Pseudomonas fluorescens BBc6R8 and/or the fungal strain Laccaria bicolor S238N on seedling growth and on the indigenous bacterial and ectomycorrhizal communities using quantitative and qualitative approaches. The field persistence of the inoculated strains was also monitored. The seedling shoot volume estimate was statistically significantly higher in the fungal inoculated plots in comparison to the non-inoculated plots but no treatment-related changes in the quantitave or qualitative microbial measurements were observed and the inoculated strains could not be detected after 4 years.     

Influence of temperature on the motility of Pseudomonas oryzihabitans and control of Globodera rostochiensis

The motility and efficacy of Pseudomonas oryzihabitans as a biocontrol agent against the potato cyst nematode Globodera rostochiensis were studied with respect to temperature. The influence of soil moisture on bacterial movement was also tested. In a closed container trial, P. oryzihabitans significantly reduced invasion of second stage juveniles (J2) of G. rostochiensis in potato roots, its effect being more marked at 25 and 21 °C than at 17 °C. P. oryzihabitans motility in vitro was optimal at 26 °C and inhibited at temperatures below 18 °C. In soil, both temperature and matric potential affected bacterial movement. At 16 °C its movement and survival were suppressed, but they were unaffected at 25 °C. At both temperatures the biocontrol agent moved faster in the wetter (−0.03 MPa) than in the drier soil (−0.1 MPa). These results suggest that temperature is a key factor in determining the potential of P. oryzihabitans as a biocontrol agent.     

节杆菌与施氏假单胞菌对甲基对硫磷的共降解研究

为实现对有机磷农药甲基对硫磷的彻底降解,研究利用有机磷农药降解菌施氏假单胞菌YC-YH1与对硝基苯酚降解菌CN2对甲基对硫磷进行共降解试验,同时克隆了参与降解过程的相关基因,并对2株菌株在对甲基对硫磷污染土壤的原位修复中的效果进行研究。结果表明,与YC-YH1单独降解相比,YC-YH1和CN2共降解在处理前32 h无较大差异;但之后,甲基对硫磷和对硝基苯酚的降解速度明显加快,处理48 h后甲基对硫磷即被完全降解,比单独用YC-H1降解时提前了16 h,处理64 h时对硝基苯酚就被完全降解。而土壤修复的研究结果表明,菌株YC-YH1与CN2能够高效降解甲基对硫磷,72 h对土壤中100 mg/kg甲基对硫磷的降解率接近100%,尤以未灭菌土壤中降解效果更好。     

Soil bacterial community response to sulfadiazine in the soil–root zone

Sulfonamide antibiotics reach soil via manure and adversely affect microbial diversity. Clear effects of these bacteriostatic, growth‐inhibiting antibiotics occur in the presence of a parallel input of microbial activity stimulating manure. Natural hot spots with already increased soil microbial activity are located in the rhizosphere, comprising microorganism such as Pseudomonas with plant growth promoting and pathogenic strains. The hypothesis was therefore that the antibiotic activity of sulfonamides is promoted in the rhizosphere even in the absence of manure, followed by shifts of the natural plant‐specific microbial community structure. This was evaluated by a laboratory experiment with Salix fragilis L. and Zea mays L. After 40 d of incubation, sub‐areas such as non‐rhizosphere soil, rhizosphere soil and plant roots were sampled. Effects on microbial community structure were analyzed using 16S rRNA gene fragment patterns of total bacteria community and Pseudomonas. Selected exoenzymes of N‐, P‐, and C‐cycling were used to test effects on microbial functions. Compared to the factors soil sub‐area and sulfadiazine (SDZ) content, plant species had the largest influence on the bacterial community structure and soil exoenzyme activity pattern. This was also reflected by an up to 1.5‐fold higher acid phosphatase activity in samples from maize‐ compared to willow‐planted soil. We conclude that antibiotic effects on the bacterial community structures are influenced by the antibiotic concentration and root influence.