Growth of zinnia,Italian ryegrass,and alfalfa and their remediation effects in diesel oil-contaminated soils

Abstract

Our objective in this study was to compare the growth of zinnia, Italian ryegrass, and alfalfa, and their remediation effects in oil-contaminated soils. The soils were prepared by mixing 2, 4, or 8% diesel oil by weight with soil. The plant height and dry weights of shoots and roots were highest for zinnia in the 2 and 4% oil treatments, and highest for Italian ryegrass in the 8% oil treatment. The reduction ratios in soil total petroleum hydrocarbons concentration (TPH) for 3 plants were lower in the 4 and 8% oil treatments than those in the 2% treatment. The reduction ratios for Italian ryegrass and zinnia contaminated with 2, 4, and 8% diesel oil treatments were significantly higher than those for alfalfa and the non-cultivation treatment at 45?days after sowing, and there were no significant differences in reduction ratios between Italian ryegrass and zinnia. The reduction ratio of soil TPH concentration brought about by zinnia was also comparable to that of Italian ryegrass. Therefore, we conclude that zinnia shows growth and remediation effects that are equivalent to those of Italian ryegrass, in soils contaminated with less than 8% oil.     

Microbial Growth and Carbon Use Efficiency in the Rhizosphere and Root-Free Soil

Plant-microbial interactions alter C and N balance in the rhizosphere and affect the microbial carbon use efficiency (CUE)–the fundamental characteristic of microbial metabolism. Estimation of CUE in microbial hotspots with high dynamics of activity and changes of microbial physiological state from dormancy to activity is a challenge in soil microbiology. We analyzed respiratory activity, microbial DNA content and CUE by manipulation the C and nutrients availability in the soil under Beta vulgaris. All measurements were done in root-free and rhizosphere soil under steady-state conditions and during microbial growth induced by addition of glucose. Microorganisms in the rhizosphere and root-free soil differed in their CUE dynamics due to varying time delays between respiration burst and DNA increase. Constant CUE in an exponentially-growing microbial community in rhizosphere demonstrated the balanced growth. In contrast, the CUE in the root-free soil increased more than three times at the end of exponential growth and was 1.5 times higher than in the rhizosphere. Plants alter the dynamics of microbial CUE by balancing the catabolic and anabolic processes, which were decoupled in the root-free soil. The effects of N and C availability on CUE in rhizosphere and root-free soil are discussed.     

Degradation of crude oil in the rhizosphere of Sorghum bicolor

Dissipation of petroleum contaminants in the rhizosphere is likely the result of enhanced microbial degradation. Plant roots may encourage rhizosphere microbial activity through exudation of nutrients and by providing channels for increased water flow and gas diffusion. Phytoremediation of crude oil in soil was examined in this study using carefully selected plant species monitored over specific plant growth stages. Four sorghum (Sorghum bicolor L.) genotypes with differing root characteristics and levels of exudation were established in a sandy loam soil contaminated with 2700 mg crude oil/kg soil. Soils were sampled at three stages of plant growth: five leaf, flowering, and maturity. All vegetated treatments were associated with higher remediation efficiency, resulting in significantly lower total petroleum hydrocarbon concentrations than unvegetated controls. A relationship between root exudation and bioremediation efficiency was not apparent for these genotypes, although the presence of all sorghum genotypes resulted in significant removal of crude oil from the impacted soil.     

Fate of polycyclic aromatic hydrocarbons (PAH) in the rhizosphere and mycorrhizosphere of ryegrass

Polycyclic aromatic hydrocarbons (PAH) can be degraded in the rhizosphere but may also interact with vegetation by accumulation in plant tissues or adsorption on root surface. Previous studies have shown that arbuscular mycorrhizal (AM) fungi contribute to the establishment and maintenance of plants in a PAH contaminated soil. We investigated the fate of PAH in the rhizosphere and mycorrhizosphere including biodegradation, uptake and adsorption. Experiments were conducted with ryegrass inoculated or not with Glomus mosseae P2 (BEG 69) and cultivated in pots filled with soil spiked with 5 g kg⁻¹ of anthracene or with 1 g kg⁻¹ of a mixture of 8 PAH in a growth chamber. PAH were extracted from root surfaces, root and shoot tissue and rhizosphere soil and were analysed by GC-MS. In both experiments, 0.006 – 0.11‰ of the initial extractable PAH concentration were adsorbed to roots, 0.003 – 0.16‰ were found in root tissue, 0.001‰ in shoot tissue and 36 – 66% were dissipated, suggesting that the major part of PAH dissipation in rhizosphere soil was due to biodegradation or biotransformation. With mycorrhizal plants, anthracene and PAH were less adsorbed to roots and shoot tissue concentrations were lower than with non mycorrhizal plants, which could contribute to explain the beneficial effect of AM fungi on plant survival in PAH contaminated soils. This revised version was published online in June 2006 with corrections to the Cover Date.     

Populations of Aerobic Bacteria Associated with the Roots of Apple and Cherry Plants

S ummary . The incidence of bacteria on the surface of apple and cherry roots was compared over a period of 20 months with those in rhizosphere and root-free soils. Seasonal variation in total and in Gram negative bacterial populations in soils and on roots was essentially similar. The highest numbers occurred between November and March and the lowest in September. Little general stimulation of bacteria near roots was detected and, on occasion, negative values were recorded for rhizosphere effect. The results indicated that edaphic factors rather than root activity had the major influence on population levels. Fluorescent pseudomonads were low in number and this suggests either that fruit tree roots were unable to support large numbers of these bacteria or that colonization from the soil populations was irregular. However, in contrast to other bacteria, there was a marked stimulatory effect on fluorescent pseudomonads, particularly in apple rhizo-spheres where a maximum rhizosphere/soil ratio of 131 was recorded.     

玉米幼苗根际土壤微生物活性对芘污染的响应

用根际袋法土培试验研究了玉米幼苗根际与非根际土壤微生物量碳、微生物熵、代谢熵和土壤酶活性对不同芘污染水平(50、200、800mg·kg-1,记为T1、T2、T3)的响应差异。结果表明,较低浓度芘可适当的刺激玉米幼苗的生长,而较高浓度芘则抑制幼苗生长,其抑制作用随芘处理浓度的提高而增强;芘对玉米根系的影响要大于对茎叶的影响。玉米幼苗能够明显促进土壤中芘的去除。根际和非根际土壤中芘的去除率分别为56.67%-76.18%和32.64%-70.44%,根际土壤中芘的平均去除率比非根际土壤高16.06%。同处理中根际土壤芘含量显著低于非根际土壤,随着芘处理浓度的提高其差异更加显著。根际土壤微生物量碳、微生物熵、多酚氧化酶活性、脱氢酶活性和磷酸酶活性均高于非根际土壤,代谢熵低于非根际土壤,且其差异随芘处理浓度的提高而增大。在不同芘污染水平下,微生物量碳、微生物熵和脱氢酶活性根际和非根际土壤为T1T2T3,代谢熵为T3T2T1;多酚氧化酶活性根际土壤为T2T1T3,非根际土壤为T1T2T3;磷酸酶活性根际土壤为T3T1T2,非根际土壤为T1T2T3。土壤中残余芘含量与土壤微生物量碳、微生物熵、多酚氧化酶、脱氢酶和磷酸酶活性呈显著负相关,与代谢熵呈显著正相关。     

Nutrients Can Enhance the Abundance and Expression of Alkane Hydroxylase CYP153 Gene in the Rhizosphere of Ryegrass Planted in Hydrocarbon-Polluted Soil

Plant-bacteria partnership is a promising strategy for the remediation of soil and water polluted with hydrocarbons. However, the limitation of major nutrients (N, P and K) in soil affects the survival and metabolic activity of plant associated bacteria. The objective of this study was to explore the effects of nutrients on survival and metabolic activity of an alkane degrading rhizo-bacterium. Annual ryegrass (Lolium multiflorum) was grown in diesel-contaminated soil and inoculated with an alkane degrading bacterium, Pantoea sp. strain BTRH79, in greenhouse experiments. Two levels of nutrients were applied and plant growth, hydrocarbon removal, and gene abundance and expression were determined after 100 days of sowing of ryegrass. Results obtained from these experiments showed that the bacterial inoculation improved plant growth and hydrocarbon degradation and these were further enhanced by nutrients application. Maximum plant biomass production and hydrocarbon mineralization was observed by the combined use of inoculum and higher level of nutrients. The presence of nutrients in soil enhanced the colonization and metabolic activity of the inoculated bacterium in the rhizosphere. The abundance and expression of CYP153 gene in the rhizosphere of ryegrass was found to be directly associated with the level of applied nutrients. Enhanced hydrocarbon degradation was associated with the population of the inoculum bacterium, the abundance and expression of CYP153 gene in the rhizosphere of ryegrass. It is thus concluded that the combination between vegetation, inoculation with pollutant-degrading bacteria and nutrients amendment was an efficient approach to reduce hydrocarbon contamination.     

Oxidation of pipecolic acid in soils and in rhizosphere soil of different plants

Soil respiration was proportional to its total carbon content. Maximum respiratory activity occurred in garden soil, followed in descending order by chernozem soil, brown soil, and sand. The oxidation of pipecolic acid, as studied by the Warburg manometric technique, in different rhizosphere soils from four crops 7, 13 and 20 days after planting as well as from one crop grown in different soils, was consistently in all cases faster than that by the corresponding non-rhizosphere soils. The curves of the rate of oxygen consumption during pipecolic acid oxidation, by garden soil (whether rhizosphere or non-rhizosphere soil) as well as by chernozem rhizosphere soil of different plants at the three stages of plant growth studied contained two peaks (two phases), whereas in non-rhizosphere chernozem soil as well as in brown soil and sand (whether affected or not affected by plant roots) only one peak was attained in the curves of the rate of oxygen uptake. The rapidity with which pipecolic acid was oxidized in the rhizosphere soil differed from plant to plant and at different phases of plant growth, and also with the type of soil used for plant growing. The extent of pipecolic acid oxidation after the first and second (if it occurred) phases did not differ in the different soils, both rhizosphere and non-rhizosphere soil, but the rate of oxygen uptake was higher in rhizosphere than in the corresponding non-rhizosphere soil. During the first phase, oxygen uptake accounted for slightly less than one-third of the total amount of oxygen required for complete oxidation of the added pipecolic acid. About two-thirds of that total amount were taken up during the both phases of oxidation.     

蚯蚓在植物修复芘污染土壤中的作用

采用盆栽试验法,研究了蚯蚓(Pheretima hupeiensis)在植物修复芘污染土壤中的作用。结果显示,试验浓度(20.24-321.42 mg/kg) 范围内,蚯蚓活动促进了芘污染土壤中修复植物黑麦草(Lolium multiforum)黑麦草的生长,其根冠比明显增大。添加蚯蚓72 d后,种植黑麦草的土壤中芘的去除率高达60.01%-86.26%,其平均去除率(74.66%)比无蚯蚓活动的土壤-植物系统(64.55%)提高10.11%,比无植物对照组(18.24%)提高56.42%。各种生物、非生物修复因子中,植物-微生物交互作用对芘去除的平均贡献率(51.75%)最为突出,比无蚯蚓活动时(44.94%)提高6.81%。说明蚯蚓活动可强化土壤-植物系统对土壤芘污染的修复作用。     

Population dynamics ofFusarium spp. in pea rhizosphere as affected by soil amendments

In nonamended soil, vegetative growth and sporulation ofFusarium spp. were higher in the rhizosphere than in the soil. Sporulation was favoured by young plants and decreased with increasing plant age. Amendments with low C/N oil-cakes enhanced vegetative growth and sporulation in root-free soil. The extent of stimulation varied with the nature of organic matter used and the stage of its deoomposition. Sporulation was suppressed by castor cake and sawdust with urea amendments. Rhizosphere cf pea altered the effect of different amendments.     

Functional diversity of the microbial community in the rhizosphere of chickpea grown in diesel fuel-spiked soil amended with <Emphasis Type="Italic">Trichoderma ressei</Emphasis> using sole-carbon-source utilization profiles

Present study describes chickpea (Cicer arietinum) growth, microbial activity and community composition in a soil samples spiked with 0, 20 (LCD) and 80 g (HCD) diesel/kg soils, amended with Trichoderma ressei. T. ressei had stimulatory effect on the plant growth parameters as compared with un-inoculated control chickpea plant. Root length, shoot length, plant dry weight and chlorophyll content enhanced 128, 31, 46, 79%, respectively, as compared over the un-inoculated control. At LCD in the presence of T. ressei chickpea root length, shoot length, plant dry weight and chlorophyll content was maximum indicating that at this concentration of diesel chickpea plants could grow very well and T. ressei amendment had synergistic effect. Effect on microbial population was most evident at HCD and resulted in 4.84 log unit reduction of heterogeneous bacterial population, as compared with LCD which caused reduction of 2.8 log unit, compared with non-diesel spiked control soil. Impact of diesel on soil was somewhat lessened in the presence of T. ressei. Our results indicated that application of diesel improved the organic matter status of soils which was in turn reflected in the higher dehydrogenase activity. This could be due to diesel being a good source of hydrocarbon readily available for microbial activity. The structure of the microbial community in rhizosphere was analyzed through the sole-carbon-source utilization profiles using ECO Biolog microplates. Significant differences were found among the diversity and evenness indices on effect of diesel on chickpea rhizosphere microflora in presence and absence of T. ressei, based on Tukey’s test (at P = 0.05). Principal component analysis of substrate source utilization pattern on Biolog Eco plates by chickpea rhizosphere microflora in presence and absence of T. ressei was determined. Distinct resolution of soil microbial communities in the presence of either diesel or, T. ressei observed thus revealed differences in the microbial metabolic profiles for the different treatments. Our results demonstrated that characteristics of the dynamics in microbial communities complemented well with organic matter status of soils and dehydrogenase activity. The technique highlighted the usefulness of this parameter for ecological indication of land use change in diesel contaminated ecosystems.     

Relationships between plant roots,proteolytic organisms and activity of protease

Proteolytic bacteria represented 18–58% of the bacterial population isolated from the rhizoplane of different crops. The activity of protease was considerably higher on roots of wheat growing in the soil than in the rhizosphere or free soil. However, only a slightly positive rhizosphere effect in the relative occurrence of casein-hydrolyzing bacteria could be observed. An indirect relationship between numbers of bacteria hydrolyzing casein and the activity of the enzyme could be found. The activity of protease related to a unit of culturable proteolytic bacteria was considerably higher on the root than in the rhizosphere and in the soil. A relationship between characteristics of the production of the enzyme by proteolytic bacteria and the protease activity on the surface of roots was demonstrated. The resulting enzyme activity on the surface of roots depended apparently on growth conditions of the plant and nature of root exudates and was influenced both by inactivation and protection due to adsorption of the enzyme by roots.     

Comparative identification by fatty acid analysis of soil,rhizosphere, and geocarposphere bacteria of peanut (Arachis hypogaea L.)

Bacterial isolates were collected from the geocarposphere, rhizosphere, and root-free soil of field grown peanut (Arachis hypogaea L.) at three sample dates, and the isolates were identified by analysis of fatty acid methyl-esters to determine if qualitative differences exist among the bacterial microflora of these zones. Five bacterial genera were associated with isolates from soil, while pod and root isolates constituted 16 and 13 genera, respectively, indicating that bacterial diversity was higher in the rhizosphere and geocarposphere than in soil. The dominant (most frequently identified) genus across all three samples dates was Flavobacterium, for pods, Pseudomonas for roots, and Bacillus, for root-free soil. Sixteen bacterial taxa were only isolated from the geocarposphere, 7 only from the rhizosphere, and 5 only from soil. These results show that specific bacterial taxa are preferentially adapted to colonization of the geocarposphere and suggest that the soil, rhizosphere, and geocarposphere constitute three distinct ecological niches. Bacteria which colonize the geocarposphere should be examined as potential biological control agents for pod-invading fungi such as the toxigenic strains of Aspergillus flavus and A. parasiticus.     

Accelerated Mineralization of Two Organophosphate Insecticides in the Rhizosphere

Numerous xenobiotic compounds, including the organophosphate insecticides O, O-diethyl-O-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate (diazinon) and O, O-diethyl-O-p-nitrophenyl phosphorothioate (parathion), appear to be degraded in the soil environment by an initial cometabolic attack. Comparing the mineralization rates of radiolabeled diazinon and parathion in root-free and in rhizosphere soil, we tested our hypothesis that, because of the presence of root exudates, the rhizosphere is an especially favorable environment for such co-metabolic transformations. The insecticides were added individually at 5 μg/g to sealed flasks containing either soil permeated by the root system of a bush bean plant or identical soil without roots. Periodically, the flask atmospheres were flushed through traps and the evolved ¹⁴CO₂ was quantitated. Bush bean plant roots without associated rhizosphere microorganisms failed to produce a significant amount of ¹⁴CO₂. During 1 month of incubation, rhizosphere flasks mineralized 12.9 and 17.9% of the added diazinon and parathion radiocarbon, respectively, compared to 5.0 and 7.8% by the soil without roots. The mineralization of parathion but not of diazinon was stimulated in a similar manner when soil without roots was repeatedly irrigated with a root exudate produced in aseptic solution culture. Viable counts of microorganisms on soil extract agar were not significantly altered by root permeation or by root exudate treatment of the soil, leaving population selection and/or enhanced cometabolic activity as the most plausible interpretations for the observed stimulatory effects. Rhizosphere interactions may substantially shorten the predicted half-lives of some xenobiotic compounds in soil.     

Enhanced remediation of chlorpyrifos by ryegrass (Lolium multiflorum) and a chlorpyrifos degrading bacterial endophyte Mezorhizobium sp. HN3

For effective remediation of contaminants, plant-endophyte partnership is a promising field to be explored. Generally endophytic bacteria assist their host plant by withstanding the stress induced by the contaminants. The objective of this study was to explore the suitability of plant-bacterial partnership for chlorpyrifos (CP) remediation using ryegrass and a CP degrading endophyte, Mesorhizobium sp. HN3 which belongs to plant growth promoting rhizobia. The inoculated yfp-tagged Mesorhizobium sp. HN3 efficiently colonized in the rhizosphere, enhanced plant growth and degradation of CP and its metabolite 3,5,6 trichloro-2-pyridinol (TCP). Significantly lower CP residues were observed in the roots and shoots of plants vegetated in inoculated soil which might be attributed to the efficient root colonization of HN3yfp. These results suggest the involvement of Mesorhizobium sp. HN3yfp in CP degradation inside the roots and rhizosphere of plants and further emphasize on the effectiveness of endophytic bacteria in stimulating the remediation of pesticide contaminants. This is the first report which demonstrates the efficacy of bacterial endophyte for degradation of CP residues taken up by the plant and enhanced remediation of chlorpyrifos contaminated soil.     

Colonization of plant rhizosphere by actinomycetes of different genera

The survival of environmental isolates of actinomycetes introduced with the seeds of agricultural plants in the root-free soil and in the rhizosphere and rhizoplane was studied. Different strategies of colonization of the rhizosphere were revealed for the representatives of the genera Streptomyces, Micromonospora, and Streptosporangium, the organisms typical for the moderate climate rhizosphere. The plants of winter rye (Secale cereale L.) inoculated with actinomycetes were shown to have growth advantages, while the cow clover plants (Trifolium pratense L.) had no growth advantages compared to uninoculated plants. The role of the plant component in the interaction with mycelial prokaryotes is discussed.     

Immobilization and mobilization of labelled sulphur in relation to soil arylsulphatase activity in rhizosphere soil of field-grown rape,barley and fallow

During plant growth, rhizosphere soils from fallow, barley (Hordeum vulgare L. cv Esterel) and rape (Brassica napus L. cv Capitole) grown in a calcareous soil were sampled 5 times (every fortnight) from May to July 2001 at plant maturity. In order to estimate the impact of C derived from photosynthesis, the aerial parts of rape and barley in an area of 1 m² were cut off about 2 cm from the soil surface, and left a fortnight before each sampling. Both soil arylsulphatase activity and a 1-week immobilization of S fertilizer in the sampled soils were then measured. The immobilization of S fertilizer was higher in fallow, followed by barley and rape rhizosphere soil. A strong positive linear correlation (r ²=0.71, P<0.001) was found between soil arylsulphatase activity and S fertilizer immobilized. Conversely, the mobilization of endogenous organic ³⁵S (obtained after leaching free and adsorbed SO₄ ^2–-³⁵S by 0.009 M Ca(H₂PO₄)₂) in the rhizosphere soil of each plant cover pooled at the end of the 5 samplings and materialized by ryegrass (Lolium perenne L. cv Massa) ³⁵S uptake, was about 3 and 2 times higher, respectively, in rape and barley than in fallow rhizosphere soil. Accordingly, strong inverse polynomial relationships were observed between soil arylsulphatase activity and ³⁵S uptake by the whole plant (r ²=0.904, P<0.02) and roots (r ²=0.970, P<0.01) of ryegrass. Plant cuttings affected both the immobilization and mobilization of S. It is concluded that the turnover of S freshly immobilized in rape rhizosphere soil was relatively high. Therefore, rape as a preceding crop in the rotations may have a beneficial effect by increasing S availability on the succeeding crop.     

Comparison of the chemical changes in the rhizosphere of the nickel hyperaccumulator Alyssum murale with the non-accumulator Raphanus sativus

Changes in pH and redox potential were studied in the rhizosphere soil of a nickel hyperaccumulator plant (Alyssum murale) and of a crop plant, radish (Raphanus sativus). Differences in rhizosphere pH and reducing activity were found between the lateral and the main roots of both species, but the pH changes in the rhizosphere were similar in both species. Changes in pH were associated with the relative uptakes of cations and anions; whether the concentrations of heavy metals in the growth medium did not have any effect on the rhizosphere pH. The source of nitrogen (ammonium or nitrate) was the major factor determining the pH of the rhizosphere of both species. The redox potential of the rhizosphere was influenced by both the N-source and the concentrations of heavy metals. When heavy metals were not present in the growth medium, and nitrate was the N-source, the reducing capacity of A. murale roots was enhanced. However, the reducing activity of A. murale was always smaller than that of radish. Therefore, the mechanism of metal solubilization by the hyperaccumulator plant does not involve either the reduction of pH in the rhizosphere or the release of reductants from roots. The acidification and reducing activity of the roots of A. murale was always smaller than that of R. sativus.     

施氮量对冬小麦根际土壤酶活性的影响

在大田高产条件下,研究了不同施氮水平对大穗型小麦品种兰考矮早八和多穗型小麦品种豫麦49-198根际土壤酶活性的影响.结果表明:两种穗型冬小麦品种根际土壤酶活性随生育进程的变化趋势一致,即蛋白酶、脲酶及脱氢酶活性均呈先升后降的变化趋势;过氧化氢酶活性随生育进程的推进逐渐增加,在成熟期达到最大值.在同生育时期内,随着施氮水平的提高,土壤蛋白酶、过氧化氢酶及脱氢酶活性均呈先增后降的变化趋势,以180 kg N·hm-2施氮水平的活性最高;脲酶活性则随施氮水平的提高而上升,在360 kg N·hm-2施氮水平下达到最高.