Promotion of plant growth and in situ degradation of phenol by an engineered Pseudomonas fluorescens strain in different contaminated environments

We show that Pseudomonas fluorescens strain P13, a plant growth-promoting bacterium, enhanced the growth of corn in uncontaminated soil but not in contaminated soil, perhaps because of its inability to reduce phytotoxicity. Another bacterial strain, Pseudomonas aeruginosa strain SZH16, showed in situ phenol-degrading activity and contained a plasmid loaded with a gene encoding for catechol 2, 3-dioxygenase, an important enzyme in the degradation pathway of aromatic compounds. We implanted this biodegradation ability into strain P13, using horizontal gene transfer techniques using strain SZH16 as the donor and P13 as the recipient, to generate a phenol-degrading transconjugant which obtained the effective plasmid from strain SZH16. Introduction of the transconjugant P13 strain into an artificially phenol-spiked soil promoted the growth of corn and in situ phenol degradation, and the increase in plant biomass correlated with the decrease in soil phenol content. Furthermore, the transconjugant P13 strain was also found to stimulate corn growth and reduce phenol concentration in water containing phenol and in historically contaminated field soils, indicating that the transconjugant strain could promote plant growth in both contaminated and uncontaminated environments. The transconjugant P13 strain was more efficient than either strain P13 or SZH16, and shows how plant growth-promoting bacteria which show no, or only limited, ability to degrade organic pollutants may be modified. This technique is attractive for many environmental remediation and agronomic applications.     

Predation by protozoa on Escherichia coli K12 in soil and transfer of resistance plasmid RP4 to indigenous bacteria in soil

Escherichia coli K12 strain (J5-3/RP4) persisted in sandy loam for more than 70 days when incubated at 10°C or 4°C. It decreased to below the level of detection within 20 days when incubated at 25°C. No loss of multi-resistance plasmid RP4 from the E. coli cells was detected during incubation in soil. There was a positive relation between the bacterial inoculum size and the following increase of the protozoan numbers in the soil. When soil microcosms were amended with an eukaryotic inhibitor, the period of survival was increased. These observations indicate a direct involvement of protozoa in the decline of E. coli in soil. Transfer of plasmid RP4 from E. coli donor bacteria to indigenous bacteria in soil was detected already 24 h after addition of the E. coli K12 donor strain. The efficiency of transfer during the first 48 h was approximately 10⁻⁶ transconjugants per donor. Inhibition of protozoan predation increased the number of transconjugants appearing in the soil, but the transfer efficiency per donor was not affected by the decreased predation. No transfer could be detected when the donor strain was washed and resuspended in saline before addition to the soil, but transconjugants were detected in this experiment when nutrients (LB) were supplemented after two days of incubation. Plasmid RP4 was maintained in the transconjugant soil bacteria throughout the experiment. The data presented here indicate that the indigenous bacteria in soil may serve as a sink for plasmidborne traits.     

Method to determine effect of antibiotics at residue levels on R-factor transfer

An analytical system was developed which can assess the ability of antibiotic/antimicrobial residues (0.01-1.00 ppm) to affect the conjugal transfer of resistance among the Enterobacteriaceae. The donor strain, Escherichia coli RP-4 (Amr Tcr Nmr Kmr Lac+), and recipient strain, E. coli Sc-8632 (Smr Lac-), were incubated together in a 1:9 donor:recipient ratio for 18 h with gentle shaking (50 rpm) in brain heart infusion broth in the presence of residue levels of antibiotics. The mating cultures were serially diluted and spread-plated onto MacConkey agar containing 25 micrograms streptomycin/mL to select the total recipient population of sensitive E. coli Sc-8632 and transconjugants. After an 18 h incubation at 37 degrees C, the plates were replicated onto MacConkey agar containing 25 micrograms ampicillin/mL to select the ampicillin-resistant transconjugant population. Repeatability was good; the average transfer was 51.8%, with a coefficient of variation of 9.3%. Residue levels of tylosin (0.10 and 1.00 ppm) increased the transfer of the ampicillin marker beyond the 95% confidence limits. Oxytetracycline, bacitracin, streptomycin, penicillin, and virginiamycin did not increase the percent transfer. Oxytetracycline at 0.01 ppm decreased the percent transfer. In general, residue levels of antibiotics (0.01-1.00 ppm) did not affect the conjugal transfer of antibiotic resistance.     

Transfer and loss of naturally-occurring plasmids among isolates of Rhizobium leguminosarum bv. viciae in heavy metal contaminated soils

Plasmid transfer among isolates of Rhizobium leguminosarum bv. viciae in heavy metal contaminated soils from a long-term experiment in Braunschweig, Germany, was investigated under laboratory conditions. Three replicate samples each of four sterilized soils with total Zn contents of 54, 104, 208 and 340 mg kg⁻¹ were inoculated with an equal number (1×10⁵ cells g⁻¹ soil) of seven different, well-characterized isolates of R. leguminosarum bv. viciae. Four of the isolates were from an uncontaminated control plot (total Zn 54 mg kg⁻¹) and three were from a metal-contaminated plot (total Zn 340 mg kg⁻¹).After 1 year the population size was between 10⁶ and 10⁷ g⁻¹ soil, and remained at this level in all but the most contaminated soil. In the soil from the most contaminated plot no initial increase in rhizobial numbers was seen, and the population declined after 1 year to <30 cells g⁻¹ soil after 4 years. One isolate originally from uncontaminated soil that had five large plasmids (no. 2-8-27) was the most abundant type re-isolated from all of the soils. Isolates originally from the metal-contaminated soils were only recovered in the most contaminated soil. After 1 year, four isolates with plasmid profiles distinct from those inoculated into the soils were recovered. One isolate in the control soil appeared to have lost a plasmid. Three isolates from heavy metal contaminated soils (one isolate from the soil with total Zn 208 mg kg⁻¹ and two isolates from the soil with total Zn 340 mg kg⁻¹) had all acquired one plasmid. Plasmid transfer was confirmed using the distinct ITS-RFLP types of the isolates and DNA hybridization using probes specific to the transferred plasmid. The transconjugant of 2-8-27 which had gained a plasmid was found in one replicate after 2 years of the most contaminated soil but comprised more than 50% of the isolates. A similar type appeared in a separate replicate of the most contaminated soil after 3 years and persisted in both of these soils until the final sampling after 4 years. After 2 years isolates were recovered from four of the soil replicates with the chromosomal type of 2-8-27 which appeared to have lost one plasmid, but these were not recovered subsequently.Isolate 2-8-27 was among the isolates most sensitive to Zn in laboratory assays, whereas isolate 7-13-1 showed greater zinc tolerance. Acquisition of the plasmid conferred enhanced Zn tolerance to the recipients, but transconjugant isolates were not as metal tolerant as 7-13-1, the putative donor. Laboratory matings between 2-8-27 and 7-13-1 in the presence of Zn resulted in the conjugal transfer of the same small plasmid from 7-13-1 to isolate 2-8-27 and the transconjugant had enhanced metal tolerance. Our results show that transfer of naturally-occurring plasmids among rhizobial strains is stimulated by increased metal concentrations in soil. We further demonstrate that the transfer of naturally-occurring plasmids is important in conferring enhanced tolerance to elevated zinc concentrations in rhizobia.     

Survival and Plasmid Transfer Ability of Escherichia Coli in Wastewater

No differences were observed in the survival of plasmid-bearingand plasmid-free Escherichia coli strains in the course of a long-term survival process in wastewater, under both illuminated and non-illuminated conditions. While the CFU counts and the number of active cells decreased, the numberof nucleoid-containing cells remained constant throughout the 30 days of experimentation. Visible light efficiently contributed to the reduction in culturability, and T₉₀ values were very different under illuminated and nonilluminatedconditions. In the latter case the time necessary to reduce theculturability of a bacterial population by 90% was 27 days, while in the former it was only 1 day. Plasmid transfer was abundant, while the survival of donor and recipient cells was extensive. After 24 hr of survival in wastewater, transfer frequency values ranged from 5.92 × 10^-5 to 1.12 × 10^-2, depending on mating conditions. In the absence of illumination, the potential transfer abilitiesremained for survival periods of at least 20 days. Transfer assays between free and adhered cells were carried out by means of dialysis bags and submerged membrane diffusion chambers. Transfer frequency for adhered cells was greater than for free cells (2.80 × 10^-2 as opposed to 2.39 × 10^-3).     

Enhanced bioremediation of soil containing 2,4-dinitrotoluene by a genetically modified Sinorhizobium meliloti

The symbiotic nitrogen-fixing soil bacterium, Sinorhizobium meliloti, is well known for its ability to interact with the leguminous plant Medicago sativa L. It has, however, not been reported that this species possesses the capability to degrade toxic nitroaromatic compounds, such as 2,4-dinitrotoluene (DNT) which is commonly associated with the degradation of the explosive trinitrotoluene (TNT). In this study, the pJS1 DNT-biodegradative plasmid was genetically transferred to S. meliloti strain USDA 1936, which was confirmed by plasmid profile analysis. Several standard analytical and chemical tests including high performance liquid chromatography (HPLC), nitrite (NO₂) release assays, rhizosphere population and plant greenhouse studies were conducted to test the ability of S. meliloti to degrade 2,4-DNT. The possible presence of 2,4-DNT remaining in the treated soil was tested, and no 2,4-DNT had been absorbed by the soil. The pJS1-carrying recombinant strain DHK1 produced ‘ARC’ alfalfa plants that were almost 2-fold higher in shoot dry weight than that produced by the parent strain on soil containing 0.14 mM 2,4-DNT. The transconjugant strain DHK1 reduced significantly one-third more 2,4-DNT in both 0.14 and 0.28 mM contaminated soil, and in 0.55 mM contaminated soil it degraded 94% of the 2,4-DNT present. In liquid cultures, however, only about 4% reduction in 2,4-DNT concentrations was obtained in 10 days. We interpret the results as clearly establishing that genetic modification was successfully used, for the first time, to improve the capability of the symbiotic nitrogen-fixing soil bacterium S. meliloti DHK1 to bioremediate in situ 2,4-DNT-contaminated soil in the presence of alfalfa plants.     

Potential of willow and its genetically engineered associated bacteria to remediate mixed Cd and toluene contamination

Purpose

The purpose of this study was to investigate if bacteria with beneficial properties that were isolated from willow growing on a metal-contaminated site can be further equipped with genes coding for a specific degradation pathway to finally obtain transconjugants that can be inoculated in willow to improve phytoremediation efficiency of mixed contaminations.

Materials and methods

Cultivable rhizosphere bacteria and root endophytes were isolated from willow (cv. Tora) growing on a metal-contaminated soil. All isolated strains were tested for their metal resistance and potential to promote plant growth. The two most promising strains were selected and were equipped with the pTOM plasmid coding for toluene degradation. Both transconjugants were inoculated separately and combined in willow cuttings exposed to mixed Cd–toluene contamination, and their effect on phytotoxicity, Cd uptake, and toluene evapotranspiration was evaluated.

Results and discussion

Many of the isolated strains tested positive for the production of siderophores, organic acids, and indole acetic acid (IAA) and showed increased Cd resistance. The Cd-resistant, siderophore-producing rhizosphere strain Burkholderia sp. HU001 and the Cd-resistant root endophyte Pseudomonas sp. HU002, able to produce siderophores, organic acids, and IAA, were selected as receptors for conjugation with the toluene-degrading Burkholderia vietnamiensis BU61 as a donor of the pTOM-TCE plasmid. Although inoculation with the individual transconjugant strains had no effect on plant growth and negatively affected Cd uptake, their combined inoculation resulted in an increased shoot biomass upon Cd–toluene exposure did not affect Cd uptake and strongly reduced evapotranspiration of toluene to the atmosphere.

Conclusions

In this study, inoculation of willow with a consortium of plant-associated bacteria equipped with the appropriate characteristics resulted in an improved phytoremediation of a mixed Cd–toluene contamination: the degradation of toluene was improved leading to a decreased toxicity and evapotranspiration, while Cd uptake and translocation were not affected.     

Factors affecting transfer of degradative plasmids between bacteria in soils

Horizontal gene transfer is useful for enhancing bioremediation through gene bioaugmentation. However, factors affecting transfer of degradative plasmids have not been systematically addressed. To this end, plasmid transfer experiments were performed using a TOL-like plasmid carrying the gene encoding for catechol 2,3-dioxygenase (C23O) between two soil bacteria under different conditions. Transfer frequency increased with air temperature in the range of 10–35 °C and reached 6 × 10⁻⁴ transconjugants per donor cell at 35 °C. The transfer frequency detected at soil depth 5–10 cm was significantly higher (p < 0.05) compared with other depths. Addition of 5–75% LB in the microbial inoculum promoted plasmid transfer frequencies. Addition of phenol to the experimental system resulted in significantly higher transfer frequency (p < 0.05) compared with no addition. Transfer frequency heat-moisture in loam was significantly higher (p < 0.05) than in other soils. The highest transfer frequency was found in the experiment containing tomato seedlings, with up to about 1.3 × 10⁻³ transconjugants per donor cell. Corn and wheat seedlings also led to significantly higher transfer frequencies (p < 0.05) compared with no plants. Furthermore, C23O activities of transconjugants formed under different conditions were measured, as a surrogate measure of the activity of transconjugant. Transfer temperature, soil and plant types had a minor influence on activities of transconjugants. Topsoil (0–5 cm) transconjugants expressed C23O more efficiently under normal incubation condition, but less efficiently when soils incubated with excessive LB medium concentrations, and in the absence of phenol in soil. These results suggested that transfer temperature, soil depth, dilutions of LB broth, phenol content, and soil and plant types had important effects on transfer of the TOL-like plasmid in soil, and some factors also affected activities of transconjugants.     

Survival of the biocontrol agents Coniothyrium minitans and Bacillus subtilis MBI 600 introduced into pasteurised, sterilised and non-sterile soils

The biocontrol agents Coniothyrium minitans and Bacillus subtilis MBI 600 were added separately to three soil types that had been either sterilised, pasteurised or left non-sterile. Applied as a conidial suspension of 1×10⁶ cfu g⁻¹ soil, C. minitans showed good survival in all sterilised, pasteurised and non-sterile soils, remaining at the numerical level at which it was applied for the duration of the 30 d experiment. Applied at a lower rate of 1×10³ cfu g⁻¹ soil, C. minitans proliferated in sterilised soil to numbers slightly over 1×10⁶ cfu g⁻¹ soil, whereas no increase was seen in pasteurised or non-sterile soils from this lower application rate. However, although C. minitans was not easily recovered on plates from non-sterile soil, it did survive at the lower numerical level in pasteurised soil, and was recoverable throughout the experiment at the rate at which it was applied. B. subtilis MBI 600 survived well following introduction as a cell suspension into sterilised soil at a rate of 1×10⁶ cfu g⁻¹ soil. Spores were formed rapidly and, after 14 d, the introduced microorganism survived in this form rather than as vegetative cells. However, in non-sterile soil, the introduced microorganism did not compete well and decreased in number, with spores being formed in low numbers. Survival of B. subtilis MBI 600 in pasteurised soil was variable, but resembled the survival seen in non-sterile soil more than that seen in sterilised soil. More B. subtilis MBI 600 spores were formed in pasteurised soil than in non-sterile soil, however, and may have been important for survival in pasteurised soil. In conclusion, this work has shown that the biocontrol agent C. minitans can survive well in soil irrespective of whether the soil has been pasteurised or not and shows good promise as a soil inoculant for control of Sclerotinia sclerotiorum. Although soil pasteurisation does improve establishment of B. subtilis MBI 600 compared to non-sterile soil, survival is relatively poor when applied as cells. The best survival of B. subtilis MBI 600 occurred as spores in sterilised soil, and spore applications to pasteurised soil in an integrated control strategy may allow sufficient establishment of the biocontrol agent to target pathogens causing damping-off.     

Survival of myxobacter strain 8 in natural soil in the presence and absence of host cells

Myxobacter strain 8 is one component of a sequence of three predatory bacteria that develop in soil when Micrococcus luteus host cells are added to the soil. The survival of strain 8 in the presence and absence of added host cells in natural soil not allowed to dry out was examined. Strain 8 vegetative cells died relatively rapidly in unamended soil. Death was faster and occurred to a greater extent in acidic than in neutral pH soil. However, in both cases death was accompanied by formation of sonication-resistant myxospores so that they comprised the ultimate population. These myxospores survived for prolonged periods in both acidic and neutral pH soils.Vegetative cells added in high numbers to soil did not multiply under any of the conditions tested. They did multiply, however, when they were added in low numbers to soil (including acidic soil) receiving sequential (additive) amendments of heart infusion broth or living M. luteus cells. This multiplication produced strain 8 cell numbers approximating those in the above experiments receiving high strain 8 cell number inoculations. Possibly, this represents a maximum vegetative cell number for soil.Germination of the myxospores in soil, followed by growth, seemed to require an approximately neutral pH and the presence of a proper host organism. Germination occurred with M. luteus as host, but not with Escherichia coli. A delayed germination occurred when sequential amendments of heart infusion broth, instead of M. luteus host cells, were made, but this could reflect a growth response by some indigenous components of the soil microflora that then served as host cells for germination.     

Impact of Fly Ash Amendment and Incorporation Method on Hydraulic Properties of a Sandy Soil

Coal fly ash has physical and chemical characteristics that makeit useful as a soil amendment, one of the more important beingthe potential to permanently improve the soil water relations ofsandy, drought-prone soils. We axemined changes in theinfiltration rate and water holding capacity of a sandy soilafter application of high rates (up to 950 Mg ha^-1) of aClass F fly ash. Fly ash was applied to large field plots byeither conventional tillage (CT; moldboard plow-disk) orintensive tillage (IT; chisel plow-rotovate-disk), and tomicroplots using a rototiller. Infiltration rate (i) wasmeasured in both studies with a disk permeameter on threeoccasions over a 12-month period. Ash effects on gravimetric water content (θ_g) at the 0–40 cm soil depth were measuredduring a 168 hr period following a 2.5 cm rainfall event andwater release curves (33 to 500 kPa) were constructed in thelaboratory using soils from the large plots. In both studiesi was decreased by ～80% one year after additionof fly ash and θ_gin ash-amended soil was higher than unamended soil throughoutthe 168 hr monitoring period. Soil water distribution variedwith tillage; the IT treatment had the highest θ_g increasesin the 0–20 cm depth while the CT treatment had θ_gincreases throughout the 0–40 cm depth. Soil water content anddistribution in ash-amended microplots were similar to ITtreatments. Fly ash amendment not only increased water holdingcapacity but also increased plant available water by 7–13% inthe 100–300 kPa range. These results suggest fly ash amendmentmay have the potential to improve crop production in excessivelydrained soils by decreasing i and increasing the amountof plant available water in the root zone.     

采用植物化感作用与诱捕作物消除列当土壤种子库

研究证明化感作用需要满足如下4个方面:1)供体植物释放化感物质使得受体植物受到连续和定量的影响;2)能从供体植物中分离、鉴定得到化感物质,而且这些化感物质无论是在室内还是在田间,都能对在自然生态系统中邻近的伴生植物产生效应;3)供体植物产生和释放的化感物质在自然条件下能以足够的浓度到达邻近受体植物;4)以足够生物活性到达受体植物的化感物质能够被吸收并能够影响受体植物的生理生化过程,而且这种影响必须排除受体植物的生长发育不是由于竞争、动物侵害、病菌感染以及物理环境等非化感因素产生的影响。列当是列当科(Orobanchaceae)列当属(Orobanche)的根寄生植物,是一种寄生于其他植物根部的全寄生植物。全世界已发现100种列当属根寄生植物,在我国危害较为严重的是向日葵列当(O.cumana)和瓜列当(O.aegyptiace)。向日葵列当主要分布在陕西北部、河北、新疆、山西、内蒙古及东北三省,主要危害向日葵。瓜列当主要分布在新疆,危害瓜类、番茄、马铃薯。列当杂草的种子体积小、重量轻(3~6μg),而且每株植物可以生产大量种子。这些种子数量巨大且在土壤中可以保持生存力长达15~20年以上。成熟后的列当种子需要经过一段时间的后熟过程,完成后熟的列当种子在发芽之前需要1~2周时间在一定的温度和湿度条件下进行预培养,预培养后的列当种子还必须从寄主那里获得一个化学物质才能发芽,在自然条件下这种发芽刺激物质是由寄主或非寄主植物的幼根分泌提供的。获得该物质后,列当种子的"发芽管"可在数日内长出种皮,之后在吸器诱导物质的作用下很快形成吸器,与寄主根吸附并穿入根内后与寄主根的木质部形成联结,从寄主植物那里竞争性地夺取水分、养分及生长激素。由于列当属植物是根寄生杂草,在没有长出地面之前,它已经给作物造成严重的伤害,所以不易控制,有效的途径是尽量减少土壤中的列当种子含量。诱捕作物是指该作物的根系能够分泌列当属植物种子发芽的刺激物质,但是又不会被列当正常寄生,诱捕作物本身可以进行正常收获。由于列当属植物种子的生命只有一次,发芽后不能寄生就会死亡,这种发芽又称之为"自杀发芽",如此可以在列当找不到寄主之前死亡,从而大大降低土壤中列当的种子库。本文介绍了作者项目组从事采用化感作用与诱捕作物(小麦、玉米、棉花、大豆等)消除列当土壤种子库最新研究进展。     

Nitrogen Status of Pollen Donor Affects Kernel Set and Yield Components in Corn

ABSTRACT

Nitrogen (N) status of corn plants influences yield performance through adjustment of yield components. Physiological function of corn pollen produced under N-stressed conditions has not drawn enough attention in genotype selection and breeding programs. The object of this study was to assess effects of N nutrition of the pollen donor on kernel set and yield components of the pollen recipient in a field trial by using a restricted pollination procedure. Pollen from plants receiving 0 and 240 kg N ha^?1 was manually applied to plants receiving 0, 80, 160, and 240 kg N ha^{? 1} during silk emergence. The high rate of N fertilizer applied to the pollen donor significantly increased kernel number, kernel weight, harvest index, and aboveground biomass in the pollen recipient. The effect of N nutrition of the pollen donor was similar at all N levels that were applied to the pollen recipient so the interaction was not significant for any of the variables.     

电融合化学激活前培养时间对绵羊重构胚发育的影响

采用绵羊（Bos gaurus）体细胞带下注入体外成熟的卵母细胞构建重构胚,比较了不同的融合和化学激活前培养时间对重构胚融合和发育的影响。融合前培养 1h,比较不同的激活前培养时间,发现各组重构胚卵裂率、桑椹胚率和囊胚率均无显著差异（P>0.05）,但激活前培养2h胚胎完整率(95.81%)高于对照（74.64%）,差异显著（P<0.05）;激活前培养2 h,比较不同的融合前培养时间,发现两组融合率,激活胚胎完整率和囊胚率无显著差异（P>0.05）,但融合前培养1h的卵裂率（76.88%）和桑椹胚率（40.63%）显著高于直接融合（64.48%和21.50%）（P<0.05）。利用G1和G2培养早期克隆胚胎,将发育至桑椹胚囊胚的胚胎移植到41只受体绵羊,仅1只妊娠但于第104天流产。用5对绵羊微卫星DNA多态性引物对该克隆流产胎儿皮肤成纤维细胞、供体细胞、受体绵羊及一只普通对照母绵羊皮肤成纤维细胞进行微卫星DNA分析,结果表明5对微卫星DNA引物扩增各组细胞DNA, 扩增产物经过10%琼脂糖凝胶电泳和银染后,条带均有明显的多态性。条带分析显示,克隆流产胎儿的微卫星DNA指纹与供体细胞完全相同,而不同于其受体母亲和对照绵羊。证明该流产羔羊基因来源于供体细胞,为体细胞克隆绵羊。     

Adsorption of pure and dirty bacterial DNA on clay minerals and their transformation frequency

Several studies have investigated the adsorption of pure DNA on soil particles and its transformation ability. However, the presence of not purified (dirty) rather than pure DNA is more common in the extracellular soil environment. For this reason, we have investigated the adsorption and binding of dirty DNA on montmorillonite (M) and kaolinite (K) and their transforming ability in comparison to pure DNA. After lysis of Bacillus subtilis cells, induced by KCl, dirty DNA was characterized by the presence of cellular wall debris (cwd) and other constitutional organic components (coc). The dirty DNA was then divided into two fractions, one with cellular wall debris (DNA +cwd) and the other without cellular wall debris (DNA −cwd). B. subtilis BD 1512 (Cm^r) and BD 170 (Cm^s) were selected as donor and recipient bacteria, respectively, for adsorption and transformation studies. Both cwd and coc seem to facilitate the adsorption of DNA to clay minerals, whereas only cwd promote the DNA binding on clays, protecting also the DNA fragments below 400 bp against nucleases. Both dirty DNA fractions adsorbed and bound on clay minerals were able to transform competent cells.     

Digestion and residue stabilization of bacterial and fungal cells,protein, peptidoglycan,and chitin by the geophagous earthworm Metaphire guillelmi

Microbial biomass is an important source of soil organic matter, which plays crucial roles in the maintenance of soil fertility and food security. However, the mineralization and transformation of microbial biomass by the dominant soil macrofauna earthworms are still unclear. We performed feeding trials with the geophagous earthworm Metaphire guillelmi using ¹⁴C-labelled bacteria (Escherichia coli and Bacillus megaterium) cells, fungal (Penicillium chrysogenum) cells, protein, peptidoglycan, and chitin. The mineralization rate of the microbial cells and cell components was significantly 1.2–4.0-fold higher in soil with the presence of M. guillelmi for seven days than in earthworm-free soil and 1–11-fold higher than in fresh earthworm cast material. When the earthworms were removed from the soil, the mineralization of the residual carbon of the microbial biomass was significantly lower than that in the earthworm-free soil, indicating that M. guillelmi affects the mineralization of the biomass in soil in two aspects: first stimulation and then reduction, which were attributed to the passage of the microbial biomass through the earthworm gut, and that the microorganisms in the cast could play only minor roles in the stimulated mineralization and residual stabilization of microbial biomass. Large amounts (8–29%) of radiolabel of the tested microbial biomass were assimilated in the earthworm tissue. Accumulation of fungal cells (11%) and cell wall component chitin (29%) in the tissue was significantly higher than that of bacterial cells (8%) and cell wall component peptidoglycan (15%). Feeding trails with ¹⁴C-lablled microbial cells and cell components provided direct evidence that microbial biomass is a food source for geophagous earthworm and fungal biomass is likely a more important food source for earthworms than bacterial biomass. Findings of this study have important implications for the roles of geophagous earthworms in the fate of microbial biomass in soil.     

A single application of Cu to field soil has long-term effects on bacterial community structure, diversity, and soil processes

Long-term diversity-disturbance responses of soil bacterial communities to copper were determined from field-soils (Spalding; South Australia) exposed to Cu in doses ranging from 0 through to 4012 mg Cu kg⁻¹ soil. Nearly 6 years after application of Cu, the structure of the total bacterial community showed change over the Cu gradient (PCR-DGGE profiling). 16S rRNA clone libraries, generated from unexposed and exposed (1003 mg Cu added kg⁻¹ soil) treatments, had significantly different taxa composition. In particular, Acidobacteria were abundant in unexposed soil but were nearly absent from the Cu-exposed sample (P<0.05), which was dominated by Firmicute bacteria (P<0.05). Analysis of community profiles of Acidobacteria, Bacillus, Pseudomonas and Sphingomonas showed significant changes in structural composition with increasing soil Cu. The diversity (Simpsons index) of the Acidobacteria community was more sensitive to increasing concentrations of CaCl-extractable soil Cu (Cu_Ext) than other groups, with decline in diversity occurring at 0.13 Cu_Ext mg kg⁻¹ soil. In contrast, diversity in the Bacillus community increased until 10.4 Cu_Ext mg kg⁻¹ soil, showing that this group was 2 orders of magnitude more resistant to Cu than Acidobacteria. Sphingomonas was the most resistant to Cu; however, this group along with Pseudomonas represented only a small percentage of total soil bacteria. Changes in bacterial community structure, but not diversity, were concomitant with a decrease in catabolic function (BioLog). Reduction in function followed a dose-response pattern with Cu_Ext levels (R²=0.86). The EC₅₀ for functional loss was 0.21 Cu_Ext mg kg⁻¹ soil, which coincided with loss of Acidobacteria diversity. The microbial responses were confirmed as being due to Cu and not shifts in soil pH (from use of CuSO₄) as parallel Zn-based field plots (ZnSO₄) were dissimilar. Changes in the diversity of most bacterial groups with soil Cu followed a unimodal response - i.e. diversity initially increased with Cu addition until a critical value was reached, whereupon it sharply decreased. These responses are indicative of the intermediate-disturbance-hypothesis, a macroecological theory that has not been widely tested in environmental microbial ecosystems.     

Carbon allocation in mycelia of arbuscular mycorrhizal fungi during colonisation of plant seedlings

The direction of carbon (C) allocation in mycorrhizal mycelia is of fundamental importance to coexistence of individual plants. We therefore investigated the transfer of C from established plants to plant seedlings through fungal mycelia. C allocation by the arbuscular mycorrhizal (AM) fungus Glomus intraradices, from ‘donor’ plants to mycelia in soil and two different species of introduced ‘receiver’ seedlings, was investigated in a pot experiment using ¹³C labelling and fatty acid analysis. After ¹³CO₂ application to the shoots of Trifolium subterraneum or Plantago lanceolata, used as donor plants, T. subterraneum and P. lanceolata receiver seedlings were introduced. Samples were collected 4-20 days after ¹³CO₂ application and analysed regarding ¹³C and the fatty acid 16:1ω5, the signature of AM fungi. ¹³C transfer from T. subterraneum donor plants was demonstrated by ¹³C enrichment of the roots of the receiver seedlings, but not from the P. lanceolata donor plants. ¹³C allocation to the neutral lipid fatty acid 16:1ω5 was only 1 ng in each receiver seedling, but 2 μg of the fatty acid in whole soil. The results indicate that C allocation through mycelial networks is influenced by the donor plant species, but is not directed towards receiver seedlings to any higher degree than towards other directions. The importance of the extraradical AM fungal mycelium as a C sink was demonstrated.     

The invasion of pea roots, pisum sativum l., by soil microorganisms, Acanthamoeba palestinensis (reich) and pseudomonas SP

Pea roots (Pisum sativum L. cultivar Tall Sugar White) were inoculated with Pseudomonas sp., isolated from the roots of Timothy grass (Phleum pratensis L.). Microscopic examination showed that the bacterium had invaded many epidermal and outer cortical cells of the root. The presence of the soil amoeba, Acanthamoeba palestinensis (Reich), which readily ingests Pseudomonas sp., did not alter the root damage. The amoebae were also found in the epidermis and outer cortex of pea roots. No amoebae were found inside pea roots when Pseudomonas sp. was absent. Bacterial invasion also occurred in pea roots grown in garden soil previously sterilized by γ-irradiation and inoculated with Pseudomonas sp., but not in the same soil inoculated and unsterilized. Timothy grass roots were similarly uninfected in inoculated unsterilized garden soil.