巨大芽胞杆菌WY4的GFP标记及其在小白菜上的定殖

解磷菌(phosphate solubilizing bacteria,PSB)可以通过提高土壤有效磷含量而增加作物产量,目前已有许多解磷菌被分离并应用于农业生产中,但关于解磷菌在植物根际中的定殖情况仍缺乏系统性的研究.WY4为本实验室前期从小白菜(Brassica chinensis)根际分离得到的一株高效解磷菌,本研究利用绿色荧光蛋白(green fluorescent protein,GFP)标记技术研究了WY4在小白菜根际及土壤中的定殖规律.与原菌株相比,GFP标记对菌株WY4-GFP生长及解磷活性具有较小影响,同时在促进小白菜生长上WY4-GFP与WY4无显著性差异;WY4-GFP具有持久的定殖能力(接种21d的自然土及30 d的小白菜根际土中,WY4-GFP的定殖数量分别为105和104 CFU/g左右),同时随时间的增加WY4-GFP在土壤中定殖数量逐渐减少;WY4-GFP在小白菜根冠及分生区大量定殖,在伸长区及侧根根毛处数量较少,同时表皮细胞间隙上也有较多的标记菌株.研究结果表明,WY4-GFP在小白菜根际及土壤中具有良好的定殖能力,这为后期深入研究解磷菌与植物间的关系提供了重要参考.     

甲基对硫磷降解菌DLLBR在青菜及根际土壤中的定殖研究

将 GFP 基因标记的甲基对硫磷降解菌 DLLBR 接入 100 ml LB 培养基,过夜培养达到 1010cfu/ml,浇灌到盆钵试验的 800 g 土壤中,20 天后用激光共聚焦显微镜检测其在小青菜植株根部和植株内的定殖及分布。结果表明标记菌株能够在植株根圈较好地定殖,也能在植株内定殖。30 天后将植株各段研磨破碎,涂布 LB 平板计数发光菌落数,在根内的定殖数为 104cfu/g 根,在茎内的定殖数为 102cfu/g 茎。结果还发现与不接菌的对照相比,处理促进了植株内细菌群落结构的变化,尤其是芽孢杆菌的种类和数量明显增加。接菌 45 天后通过土壤计数检测发现,标记菌株在土壤中的存活力很高,能达到 106cfu/g 土,同时也发现与对照相比,接菌的土壤细菌群落结构明显发生了变化,细菌种类变化不大,但芽孢杆菌的数量明显增加。     

钩状木霉 ACCC31649的GFP标记及其对辣椒定殖和促生作用

【目的】旨在建立农杆菌介导的绿色荧光蛋白 (GFP) 基因转化钩状木霉 (Trichoderma hamatum) ACCC31649的技术方法,筛选遗传稳定的GFP标记转化子,并研究该菌株在辣椒植株中定殖和对辣椒的促生作用,为进一步阐明木霉在辣椒根际定殖及其与辣椒病原菌在辣椒根际和植株中的定殖、互作和生防作用奠定基础。【方法】通过根癌农杆菌介导的遗传转化方法筛选遗传稳定的GFP标记转化子,通过灌根接种方法和组织切片的水玻片荧光观察研究了钩状木霉在辣椒植株中定殖过程和对辣椒的促生作用。【结果】获得了遗传稳定GFP标记的钩状木霉转化子。荧光显微观察表明,辣椒根、茎、叶组织中都检测到GFP标记菌株的定殖。标记菌株首先在根部定殖,然后通过根部维管束逐步定殖到茎和叶片组织中。野生型菌株和GFP标记菌株灌根接种4叶期辣椒幼苗,30天后,GFP标记菌株与水处理对照相比,辣椒的株高增长13.5%,根长增长16.2%,鲜重和干重分别增加了43.8%和45.3%,而且野生型菌株与GFP标记菌株对辣椒的促生作用没有显著差异。【结论】钩状木霉能够在辣椒植株根、茎和叶组织中定殖,并且对辣椒具有显著的促生作用。同时,GFP标记的钩状木霉将在进一步阐明该菌株在辣椒根际定殖及其对病原菌拮抗和互作研究中发挥重要作用。     

GFP标记的植物促生菌B96-Ⅱ-gfp的定殖能力研究

采用绿色荧光蛋白基因标记技术研究了植物促生菌B96-Ⅱ的标记菌B96-Ⅱ-gfp在盆栽黄瓜土壤中的时间、空间定殖动态以及在黄瓜植株上的分布。研究表明: B96-Ⅱ-gfp在土壤中具有持久的定殖能力, 接种180 d时在自然土和黄瓜枯萎菌病土中的定殖数量分别为2.7×10⁴ cfu·g^-1和6.6×10⁴ cfu·g^-1, 360 d时在土壤中仍可检测到B96-Ⅱ-gfp的存在。B96-Ⅱ-gfp可在盆栽植株生长土壤的表层(0~4 cm)、中层(4~8 cm)和底层(8~12 cm)定殖, 定殖数量随土壤深度的增加而增加。此外, B96-Ⅱ-gfp还可在黄瓜的根、茎和叶上定殖, 根部定殖的数量(7.2×10⁴ cfu·g^-1)显著高于茎部和叶部定殖的数量; 黄瓜植株体内定殖的数量多于体表定殖的数量。对土壤中可培养的3大微生物类群影响的研究表明: B96-Ⅱ-gfp对土壤中真菌数量具有显著抑制作用, 而对细菌和放线菌数量则没有明显影响。防病促生试验表明: B96-Ⅱ-gfp可增加黄瓜株高、鲜重和干重, 对黄瓜枯萎病有一定的防治效果, 且与未标记菌株B96-Ⅱ的防病促生作用无显著差异。     

短小芽孢杆菌BX-4抗生素标记及定殖效果研究

为研究短小芽孢杆菌BX-4在作物根际的定殖及防病效果,通过浓度梯度法用氨苄青霉素对其进行了抗性标记,并通过番茄盆栽试验研究了其在根际土壤中的定殖规律。结果表明,筛选出的突变体菌株BX-4＇能够耐受浓度为200 μg·mL^-1的氨苄青霉素,并且具有耐药和遗传双重稳定性;应用试验显示该突变体菌株能成功在番茄根际定殖,接种20 d后根际土壤中存活数量达到最高值1.34×10^8 cfu·g^-1干土,以后逐渐下降,到50 d时趋于稳定;筛选的突变体菌株对番茄青枯病具有明显的防治效果,防效达37.9%-50.9%。短小芽孢杆菌BX-4在作物根部的定殖规律为揭示其生防机理及应用该菌提供了科学根据。     

田菁茎瘤固氮根瘤菌对小麦种子侵染的促生作用及其在根系内的定殖

选取8个常见小麦品种,通过室内限菌试验筛选出对于田菁茎瘤固氮根瘤菌Azorhizobium caulinodans ORS571 (A.caulinodans) 侵染响应敏感的品种,研究了浸种侵染、菌液浓度和添加葡萄糖对接菌小麦幼苗生长的影响,并结合荧光显微镜检测绿色荧光蛋白（GFP）标记A.caulinodans在小麦幼苗根系内的分布与定殖规律。结果表明:小偃22为响应敏感品种;在室内限菌条件下,浸种侵染小偃22幼苗平均根长和平均株高分别较对照增加了17.04%和8.37%;最适菌液浓度为1.0108个/mL;在菌液中添加1g/L葡萄糖有助于A.caulinodans浸种侵染和定殖。荧光显微镜检测结果显示,GFP标记A.caulinodans从小麦幼苗根毛和侧根裂隙处进入,定殖于根维管组织等部位。田间试验结果进一步表明A.caulinodans浸种侵染对不同小麦品种均具较明显的促生作用。     

内生菌YN201728的定殖能力及其防治烟草白粉病的效果研究

为研究生防菌YN201728在烟草体内的定殖规律及防病机制,本研究利用其绿色荧光标记菌株YN28-P43GFPmut3a实时动态观测标记菌的定殖部位及密度,并探究其对温室烟草白粉病的盆栽防效与定殖的关系。结果表明,YN28-P43GFPmut3a发酵液处理烟草种子和幼苗后,种子内的标记菌含量可达2.18×10⁶ CFU·g^-1,在幼苗的根表土、根际土、根、茎、叶等组织中均能检测到标记菌,且其定殖密度表现为根表土>根际土>根>茎>叶。激光共聚焦显微镜观察显示,标记菌主要聚集在烟草根表皮、木质部导管、茎表皮、韧皮部及维管束组织、叶片表面和叶肉细胞间隙以及种皮与胚等部位。盆栽防效试验结果表明,YN201728野生型和标记菌株对烟草白粉病均有较好的保护和治疗效果,持效期长达21 d。此外,烟草叶片中内生菌的定殖量与其防效呈正相关。本研究结果表明,内生菌YN201728在烟草体内有良好的定殖和防治白粉病效果,具很好的开发潜力,为烟草病害的生物防治提供了一定的理论基础。     

发光酶基因(luxAB)标记的荧光假单胞菌CP1108的定殖能力研究

利用三亲本杂交方法,将luxAB发光酶基因标记至荧光假单胞菌(Pseudomonas fluorescens)CP1108上,研究标记菌株CP1108L的个体生态学特征及其在棉花根圈的定殖动态.结果表明,标记菌株连续传代20次均未发生质粒丢失现象,标记质粒在受体菌株中较为稳定,CP1108L菌株的生长及部分生理生化特性...     

解磷细菌 K3 的 GFP 标记及其解磷能力检测

本文运用构建成功的含有假单胞菌属自身启动子及绿色荧光蛋白(GFP)的质粒pTRGFP电转至假单胞菌解磷细菌K3中,通过激光共聚焦显微镜及质粒检测,获得了发光稳定的标记菌株K3GFP。7天液体摇瓶试验中,标记菌株K3GFP的可溶性P含量在第4天达到最高774μg/ml,而出发菌株K3在第3天时已经达到了最大含量780μg/ml,说明pTRGFP质粒的转入对K3菌株解P能力有一定的影响。标记菌株K3GFP施入自然土壤10天后数量维持在5.47×106CFU/g～2.40×106CFU/g,35天后降到5.0×103CFU/g,说明解磷细菌K3GFP可以在自然土壤中定殖。本试验为研究解磷细菌在根际及土壤中的生长动态等行为特征奠定了基础。     

一种初步判断细菌类微生物在土壤中定殖能力的方法

为了缩短功能微生物从筛选获得到生产利用的周期，研究一种简便的判断细菌类微生物在土壤中定殖能力的方法。基于功能菌株对 21种抗生素的敏感性计算敏感值，利用盆栽试验检测功能菌株在土壤中的定殖能力，并分析敏感值与在土壤中定殖能力间的关系。结果表明，敏感值和功能菌株在土壤中的定殖能力呈负相关，当敏感值达到 85时功能菌株在土壤中的定殖能力较差，通过功能菌株对 21种抗生素的敏感值来初步判断其定殖能力具有可行性。     

Effect of Biuret Content in Urea on the Growth and Nitrogen uptake of some Grain Crops in Egyptian Soils

Seven pot experiments were conducted to study the direct and the accumulative effect of urea containing (0.4, 1.0, 1.5, 2.0, 3.0 and 4.0 %) biuret on the growth of rice, wheat, and maize plants and their N content and N uptake in two fine textured alluvial soils and one sandy clay loam (highly calcareous) soil. It was found that both rice and wheat grain yields were not significantly reduced in all soils where urea containing high percentage of biuret was used namely 3 % and 4 %. Ammonium sulphate fertilizer was superior to urea in plant production only in the sandy clay loam. Moreover, the N recovery was higher in the alluvial soils in comparison to the sandy clay loam. The accumulative addition of biuret to the soils did not further reduce either the growth rate or the N uptake of the plants.     

Studies on soil fumigation—II: Effects on bacteria

In a South Australian wheat-field soil the viable counts of “total” aerobic bacteria and of fluorescent pseudomonads were initially greatly depressed by fumigation with 220 kg·ha^?1 chloropicrin (CP) or with a combined application of 220 kg·ha^?1 of chloropicrin and 220 kg·ha^?1 methyl bromide (F). Bacterial numbers rose sharply within 10 days of the completion of fumigation. For a further 14 days the fluorescent pseudomonads formed the major part of the aerobic bacterial population counted and over 5 months later their numbers in F-treated soil remained about 10 times higher than in untreated soil. Numbers of aerobic spore-formers rose more slowly after CP or F treatment. but then remained significantly higher over the 159 days of the trial. Fumigation with 220 kg·ha^?1 of methyl bromide alone (MB) had little effect on soil bacterial numbers.A check of random isolates revealed a predominance of Gram-negative organisms in soil treated with CP, this dominance decreasing with time, whereas MB treatment did not result in any detectable change.Fluorescent pseudomonads from rhizospheres of wheat plants in soil fumigated with CP contained smaller proportions of strains antagonistic in vitro to Gaeumannomyces graminis var. tritici than isolates from MB-treated soil or from untreated soil.     

复混肥中缩二脲含量对作物生长的影响

采用水培和盆栽法,研究了复混肥中不同含量的缩二脲对作物生长的影响.水培施肥水平为每升水溶液中复混肥(10-8-7)含量分别为0.5 g和1.0 g,即每升水溶液中含氮量达到56.05 mg和112.1 mg;土壤施肥水平为每千克土壤中复混肥(10-8-7)含量分别为0.5 g和1.0 g,即每千克土壤中含氮量达到56.05 mg和121.1 mg.水培试验结果表明:每升水溶液中,在适宜各供试作物生长的施肥条件下,复混肥中缩二脲对冬小麦、玉米、番茄、油菜、水稻的毒害作用的临界值分别为35、40、20、15、15 mg.盆栽试验结果表明:在适宜各供试作物生长的施肥条件下,缩二脲对冬小麦、玉米、番茄、油菜、水稻的毒害作用的临界值分别为所施复混肥(10-8-7)含量的1.6%、2.0%、2.0%、2.0%、2.0%.当高于这个临界值时,缩二脲对此5种作物会造成毒害作用.考虑到复混肥的实际制造过程中大量使用含氯化肥,氯离子含量波动大,建议国家在制定复混肥产品标准时缩二脲含量应限制在1%以下为宜.     

从土壤中分离获得的五氯硝基苯降解菌株降解五氯硝基苯特性研究

A bacterial strain,pcnb-21,capable of degrading pentachloronitrobenzene(PCNB) under aerobic and anoxic conditions,was isolated from a long-term PCNB-polluted soil by an enrichment culture technique and identified as Labrys portucalensis based upon its morphological,physiological and biochemical properties,as well as 16S rRNA gene sequence analysis.Effects of different factors,such as temperature and pH,on PCNB biodegradation were studied.Strain pcnb-21 efficiently degraded PCNB at temperatures from 20 to 30 ℃ and initial pH values from 4 to 7,which might be the first time that a Labrys strain was found capable of efficiently degrading PCNB.The degradation of PCNB was affected by oxygen,and the degradation decreased with increasing aeration.Exogenous electron donors such as glucose,lactic acid and succinic acid promoted the biodegradation of PCNB,while electron acceptors such as sodium nitrite,sodium sulfate,sodium nitrate and sodium sulfate inhibited PCNB biodegradation.The degradation of PCNB in sterile and non-sterile soils by a green fluorescent protein(GFP)-labeled strain,pcnb-21-gfp,was also studied.Cells of pcnb-21-gfp efficiently degraded 100 mg kg -1 PCNB in sterile and non-sterile soils and could not be detected after 42 days.Strain pcnb-21 might be useful in bioremediating PCNB-polluted soils and environment.     

Increased degradation of phenanthrene in soil by Pseudomonas sp. GF3 in the presence of wheat

A phenanthrene-degrading bacterial strain Pseudomonas sp. GF3 was examined for plant-growth promoting effects and phenanthrene removal in soil artificially contaminated with low and high levels of phenanthrene (0, 100 and 200 mg kg⁻¹) in pot experiments. Low and high phenanthrene treatments significantly decreased the growth of wheat. Inoculation with bacterial strain Pseudomonas sp. GF3 was found to increase root and shoot growth of wheat. Strain GF3 was able to degrade phenanthrene effectively in the unplanted and planted soils. Over a period of 80 days the concentration of phenanthrene in soil in which wheat was grown was significantly lower than in unplanted soil (p<0.05). At the end of the 80-d experiments, 62.2% and 42.3% of phenanthrene had disappeared from planted soils without Pseudomonas sp. GF3 when the phenanthrene was added at 100 and 200 mg kg⁻¹ soil, respectively, but 84.8% and 70.2% of phenanthrene had disappeared from planted soils with the bacterial inoculation. The presence of vegetation significantly enhances the dissipation of phenanthrene in the soil. There was no significant difference in soil polyphenol oxidase activities among the applications of 0, 100 and 200 mg kg⁻¹ of phenanthrene. However, the enzyme activities in planted and unplanted soils inoculated with the strain Pseudomonas sp. GF3 were significantly higher than those of non-inoculation controls. The bacterial isolate was also able to colonize and develop in the rhizosphere soil of wheat after inoculation.     

木醋液对滨海盐土解磷真菌Apophysomyces sp.SM-1生长代谢及其溶磷效果的影响

为缓解不溶性磷酸盐累积造成的盐碱土壤板结问题,从江苏滨海盐碱土壤中筛选到一株具有较强溶磷能力的真菌菌株SM-1,并初步鉴定为Apophysomyces sp.,同时考察了木醋液对该菌生长及溶磷效果的影响。结果表明,木醋液的添加能够显著影响菌株SM-1的生长,添加量为0.1%的木醋液是其最适宜的生长浓度。菌株SM-1对不同磷源有着不同的分解能力,其中对磷酸三钙的分解能力最佳,培养基中水溶磷含量由93 mg·L~(-1)提高到246.9 mg·L~(-1)。木醋液对供试磷源没有分解效果,但添加木醋液则能够显著提高菌株对磷矿粉的分解能力,培养基中水溶磷含量由79 mg·L~(-1)提高到115.3 mg·L~(-1),而对磷酸三钙的分解能力与单独接入菌株SM-1的效果相当。这预示着菌株SM-1与木醋液混合使用在改善盐碱化土壤肥力方面具有很好的应用前景。     

Fluctuations in bacterial populations on the root surface of wheat (Triticum aestivum L.) grown under different soil conditions

Summary Populations of several bacterial groups on the root surface of wheat and in root-free soil were investigated in volcanic ash soil and non-volcanic ash soil throughout a series of predetermined intervals. Over time, the populations changed similarly both on the root surface and in root-free soil. The numbers of total bacteria, fluorescent Pseudomonas spp., phosphate-solubilizing bacteria, and NH _inf+ ^sup4 -oxidizing bacteria, were consistently lower in the plots with volcanic ash soil than with nonvolcanic ash soil, but the numbers of cellulose-decomposing bacteria were opposite to those of the other groups. Superphosphate application improved the growth of wheat in the volvanic ash soil. It did not, however, bring about any significant changes in the bacterial populations among the volcanic ash soils supplemented with three different levels of superphosphate, though there were some variations with plant age.     

Effects of Klebsiella planticola SDF20 on soil biota and wheat growth in sandy soil

The potential for ecological effects to occur after the release of genetically engineered microorganisms is a global concern and the release of biotechnology products must be assessed on a case-by-case basis. In this research, a genetically engineered strain of Klebsiella planticola (SDF20) bacteria was added to microcosms containing sandy soil and wheat plants to assess the potential for effects on soil biota and plant growth. One half of the soil treatments in this study contained wheat plants to compare some effects on growing rhizosphere communities in the experimental system. When SDF20 was added to soil with plants, the numbers of bacterial and fungal feeding nematodes increased significantly, coinciding with death of the plants. In contrast, when the parental strain, SDF15 was added to soil with plants, only the number of bacterial feeding nematodes increased, but the plants did not die. The introduction of either SDF15 or SDF20 strains to soil without plants did not alter the nematode community. No effects were observed on the activity of native bacterial and fungal communities by either SDF15 or SDF20. This study is evidence that SDF20 can persist under conditions found in some soil ecosystems and for long enough periods of time to stimulate change in soil biota that could affect nutrient cycling processes. Further investigation is needed to determine the extent these observations may occur in situ but this study using soil microcosms was the first step in assessing potential for the release of genetically engineered microorganisms to result in ecological effects.     

草浆造纸木质素对土壤性质和小白菜生长的影响

草浆造纸废液是影响秸秆造纸发展的重要因素,利用小白菜盆栽试验研究了施用麦秸造纸废液木质素和麦秸对土壤性质及小白菜生长的影响,以期为造纸废弃物的资源化利用提供依据。结果表明,两茬小白菜收获后,铵木素配施N肥使土壤pH值降低;小白菜两茬连作后铵木质素配施的土壤活性有机碳含量及多酚氧化酶活性分别为2.73g·kg-1和1.77mg·g-1·2h-1,显著高于其他处理;除对照和单施氮肥外,两茬盆栽收获后土壤多酚氧化酶活性均高于第一茬试验。两茬小白菜收获后秸秆配施和碱木质素配施氮肥土壤过氧化物酶活性均高于其他处理。铵木质素配施及秸秆配施的小白菜干重相近,显著高于碱木质素配施。研究表明,与秸秆还田相比,施用铵木质素没有对土壤性状和小白菜生长产生不利影响,故铵法草浆木质素可以作为一种潜在的土壤有机改良剂应用。     

Carbon and net nitrogen mineralisation in two forest soils amended with different concentrations of biuret

Biuret is a known contaminant of urea fertilisers that might be useful as a slow release N fertiliser for forestry. We studied carbon (C), net nitrogen (N) mineralisation and soil microbial biomass C and N dynamics in two forest soils (a sandy loam and a silt loam) during a 16-week long incubation following application of biuret (C 23.3%, N 40.8%, O 30.0% and H 4.9%) at concentrations of 0, 2, 10, 100 and 1000 mg kg⁻¹ (oven-dried) soil to assess the potential of biuret as a slow-release N fertiliser. Lower concentrations of biuret specifically increased C mineralisation and soil microbial biomass C in the sandy loam soil, but not in the silt loam soil. A significant decrease of microbial biomass C was found in both soils at week 16 after biuret was applied at higher concentrations. C mineralisation declined with duration of incubation in both soils due to decreased C availability. Biuret at concentrations from 10 to 100 mg kg⁻¹ soil had a significantly positive priming effect on soil organic N mineralisation in both soils. The causes for the priming effects were related to the stimulation of microbial growth and activity at an early stage of the incubation and/or the death of microbes at a later stage, which was biuret-concentration-dependent. The patterns in NH₄⁺-N accumulation differed markedly between the two soils. Net N mineralisation and nitrification were much greater in the sandy loam soil than in the silt loam soil. However, the onset of net nitrification was earlier in the silt loam soil. Biuret might be a potential slow-release N source in the silt loam soil.