Isolation,characterization, and identification of bacteria associated with the zinc hyperaccumulator Thlaspi caerulescens subsp. calaminaria

We investigated bacterial populations associated with the Zn hyperaccumulator Thlaspi caerulescens subsp. calaminaria grown in a soil collected from an abandoned Zn-Pb mine and smelter in Plombières, Belgium. The bacterial population of the nonrhizospheric soil consisted of typical soil bacteria, some exhibiting multiple heavy-metal resistance characteristics that often are associated with polluted substrates: 7.8% and 4% of the population survived in the presence of elevated levels of Zn (1 mM) and Cd (0.8 mM), respectively. For the bacterial population isolated from the rhizosphere, the comparable survival rates were 88 and 78%. This observation indicates a selective enrichment of the metal-resistant strains due to an increased availability of the metals in soils near the roots compared with nonrhizospheric soil. The endophytic inhabitants of the roots and shoots were isolated, identified, and characterized. Although similar endophytic species were isolated from both compartments, those from the rhizoplane and roots showed lower resistance to Zn and Cd than the endophytic bacteria isolated from the shoots. In addition, root endophytic bacteria had additional requirements. Contrary to the rootresiding inhabitants, the shoot represented a niche rich in metal-resistant bacteria and even seemed to contain species that were exclusively abundant there. These differences in the characteristics of the bacterial microflora associated with T. caerulescens might possibly reflect altered metal speciation in the different soils and plant compartments studied.     

Soil microbial diversity as affected by the rhizosphere of the hyperaccumulator Thlaspi caerulescens under natural conditions

It is hypothesized that metal hyperaccumulator plants have specific rhizosphere conditions, potentially modifying the bioavailability of soil metals. This article aims to further the knowledge about the rhizosphere of the hyperaccumulator Thlaspi caerulescens, focusing on its microflora isolated from metalliferous soils collected in situ where the plants grow naturally. We characterized the cultivable microbial communities isolated from the rhizosphere of one population of this Ni hyperaccumulator species grown on a serpentine soil. The rhizosphere soil harbored a wide variety of microorganisms, predominantly bacteria, confirming the stimulatory effect of the T. caerulescens rhizosphere on microbial growth and proliferation. We tested the hypothesis that the rhizosphere of T. caerulescens influences (1) the metabolic diversity of the bacterial community and (2) the bacterial resistance to metals. The principal component analysis of the Biolog plate's data confirmed a structural effect of the rhizosphere of T. caerulescens on bacterial communities. The percentage of Ni-resistant bacteria was higher in the rhizosphere than in the bulk soil, suggesting a direct effect of the rhizosphere on Ni tolerance, reflecting a greater bacterial tolerance to Ni in the rhizosphere.     

Heavy metal uptake and chemical changes in the rhizosphere of Thlaspi caerulescens and Thlaspi ochroleucum grown in contaminated soils

Thlaspi caerulescens (J. and C. Presl) and Thlaspi ochroleucum (Boiss. ex Heldr) were grown in three different soils containing moderate to high amounts of heavy metals in a pot experiment, using a rhizobag technique. T. caerulescens accumulated significantly more Zn in the shoots than T. ochroleucum. The concentrations of Zn in the shoots of T. caerulescens ranged from 3100 to 8100 mg kg^-1 dry matter (DM), but only from 800 to 1600 mg kg^-1 DM in T. ochroleucum. Total uptake of Zn in the shoots of T. caerulescens was about 5 times that of T. ochroleucum. In contrast, the differences between the two species in the uptake of Cd, Cr, Cu, Ni and Pb were generally small. Concentrations of mobile Zn (extractable with 1M NH₄NO₃) in the rhizosphere and non-rhizosphere soils decreased considerably after growth of both plants, but the decreases were greater with T. caerulescens than with T. ochroleucum. The decreases in the mobile fraction accounted for less than 10% of the total Zn uptake by T. caerulescens indicating that this species was effective in mobilising Zn from less soluble fractions in the soils. The rhizosphere soils tended to have higher concentrations of mobile Zn than the non-rhizosphere soils, probably because of the lower pH in the rhizosphere. The pH in the rhizosphere soils was 0.2-0.4 units lower than that in the non-rhizosphere soils at the end of the experiment. However, there were no significant differences between the two species in the degree of rhizosphere acidification. The results suggest that T. caerulescens has potential for removing Zn from moderately to highly contaminated soils, but that this ability was not related to the pH changes in the rhizosphere.     

Phenotypic characterization of microbes in the rhizosphere of Alyssum murale

Metal hyperaccumulator plants like Alyssum murale are used for phytoremediation of Ni contaminated soils. Soil microorganisms are known to play an important role in nutrient acquisition for plants, however, little is known about the rhizosphere microorganisms of hyperaccumulators. Fresh and dry weight, and Ni and Fe concentrations in plant shoots were higher when A. murale was grown in non-sterilized compared to sterilized soils. The analysis of microbial populations in the rhizosphere of A. murale and in bulk soils demonstrated that microbial numbers were affected by the presence of the plant. Significantly higher numbers of culturable actinomycetes, bacteria and fungi were found in the rhizosphere compared to bulk soil. A higher percent of Ni-resistant bacteria were also found in the rhizosphere compared to bulk soil. Percentage of acid producing bacteria was higher among the rhizosphere isolates compared to isolates from bulk soil. However, proportions of siderophore producing and phosphate solubilizing bacteria were not affected by the presence of the plant. We hypothesize that microbes in the rhizosphere of A. murale were capable of reducing soil pH leading to an increase in metal uptake by this hyperaccumulator.     

Fatty acid methyl ester (FAME) profiles as a tool to investigate community structure of two agricultural soils

Soil microbiological parameters may be the earliest predictors of soil quality changes. Recently, molecular techniques such as fatty acid methyl ester (FAME) profiles have been used to characterize soil microbial communities. Fatty acid methyl ester (FAME) from whole soil may be derived from live cells, dead cells, humic materials, as well as plant and root exudates. Our objective was to verify differences in FAME profiles from two agricultural soils with different plants. Soil samples were collected from Ritzville and Palouse silt loams for fatty acid analysis. Soil samples from wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), pea (Pisum sativum L.), jointed goatgrass ( Aegilops cylindrica L.) and downy brome (Bromus tectorum L.) rhizospheres were also collected for fatty acid analysis. Principal component analysis (PCA) of the two soils explained 42% of the variance on PC1, which accounted for Palouse soil. Ritzville soil accounted for 19% of the variance on PC2. Factor analysis showed that rhizosphere microbial communities from various plant species may differ depending on the plant species. Presence of Gram-positive bacteria as identified by a15:0, i15:0, a17:0 and i17:0 peaks were similar between rhizosphere and nonrhizosphere soils. Gram-negative bacteria characterized by short chain hydroxy acids (10:03OH and 12:03OH) as well as cyclopropane acids (cy17:0) were higher in rhizosphere soil than nonrhizosphere. This indicates a possible shift in the bacterial community to more Gram-negative bacteria and fewer Gram-positive bacteria in the rhizospheres of the plants species studied.     

Impact of metal pollution and Thlaspi caerulescens growth on soil microbial communities

Soil microorganisms drive critical functions in plant-soil systems. As such, various microbial properties have been proposed as indicators of soil functioning, making them potentially useful in evaluating the recovery of polluted soils via phytoremediation strategies. To evaluate microbial responses to metal phytoextraction using hyperaccumulators, a microcosm experiment was carried out to study the impacts of Zn and/or Cd pollution and Thlaspi caerulescens growth on key soil microbial properties: basal respiration; substrate-induced respiration (SIR); bacterial community structure as assessed by PCR-denaturing gradient gel electrophoresis (DGGE); community sizes of total bacteria, ammonia-oxidizing bacteria, and chitin-degrading bacteria as assessed by quantitative PCR (Q-PCR); and functional gene distributions as determined by functional gene arrays (GeoChip). T. caerulescens proved to be suitable for Zn and Cd phytoextraction: shoots accumulated up to 8,211 and 1,763 mg kg(-1) (dry weight [DW]) of Zn and Cd, respectively. In general, Zn pollution led to decreased levels of basal respiration and ammonia-oxidizing bacteria, while T. caerulescens growth increased the values of substrate-induced respiration (SIR) and total bacteria. In soils polluted with 1,000 mg Zn kg(-1) and 250 mg Cd kg(-1) (DW), soil bacterial community profiles and the distribution of microbial functional genes were most affected by the presence of metals. Metal-polluted and planted soils had the highest percentage of unique genes detected via the GeoChip (35%). It was possible to track microbial responses to planting with T. caerulescens and to gain insight into the effects of metal pollution on soilborne microbial communities.     

Zinc and cadmium uptake by the hyperaccumulator Thlaspi caerulescens in contaminated soils and its effects on the concentration and chemical speciation of metals in soil solution

The hyperaccumulator Thlaspi caerulescens J & C Presl. was grown in seven different soils collected from around Europe that had been contaminated with heavy metals by industrial activity or the disposal of sewage sludge to land. Zinc accumulation factors (shoot concentration/initial soil solution concentration) ranged from 3500–85 000 with a mean value of around 36 000. This compares with mean accumulation factors of 636, 66 and 122 for Cd, Ca and Mg, respectively. The concentration of Zn in the shoots was much greater than in the roots. The total removal of Zn and Cd ranged from 8 to 30 and from 0.02 to 0.5 mg kg^-1 soil, respectively. The Zn concentration in shoots of T. caerulescens correlated, using a curvilinear relationship, with the initial Zn concentration in soil solution (R² = total Zn 0.78; Zn²+ 0.80). There was no relationship between the uptake of Zn and the total Zn concentration in the soil. In most soils, solution pH increased only slightly after growth of T. caerulescens, indicating that acidification was not the mechanism used to mobilise Zn in the soil. Dissolved organic carbon concentrations generally increased but characterisation of the component organic compounds was not attempted. The concentrations of Zn and Cd in soil solution decreased considerably after growth of T. caerulescens. The percentages of Zn and Cd in soil solution present as free ions also decreased. However, the decrease of Zn in soil solution after growth accounted for only about 1% of the total Zn uptake by T. caerulescens. This was much lower than for Cd, Ca and Mg. The results suggest that either T. caerulescens was highly efficient at mobilising Zn which was not soluble initially, or the soils used had large buffering capacities to replenish soil solution Zn within a short time. This work highlights the need to investigate the role of root exudates on the mobilisation of Zn and Cd in soils by the hyperaccumulator T. caerulescens.     

Populations of the spinach wilt pathogen, Fusarium oxysporum f. sp. spinaciae, in the root tissues, rhizosphere, and soil in the field.

Populations of Fusarium oxysporum f. sp. spinaciae in root tissues and rhizosphere soil of diseased spinach plants were higher than in the root tissues and rhizosphere soil of healthy plants. Populations in soil rhizosphere were higher than in nonrhizosphere soil. The fungus populations were very low in the root tissues of the nonsusceptible strawberry, broccoli, chinese cabbage, and mustard grown in the infested field. The populations were low at the beginning of the season, increased, and remained high during the summer, then dropped in the fall. The fungus populations ranged from 1600 to 2600 propagules/g in the top 10 cm of soil, declined sharply between 11 and 20 cm, and were nondetectable between 41 and 60 cm.     

玉米根际与非根际解磷细菌的分布特点

植物光合作用产物约有 12 %～ 5 0 %通过根系进入根际土壤中 ,不同的植物 ,同一植物不同的生长发育时期 ,不仅根际分泌物的数量有差异 ,而且分泌物的种类也不同[4 ] 。这些分泌物不仅是微生物很好的培养基 ,而且一些分泌物可能抑制或有利于甚至刺激某些微生物的繁殖 ,从而导致根际微生物种群结构的变化。根际微生物的数量、活性和群落结构及其变化 ,直接影响到植物吸收水分、养分 ,也影响植物对恶劣环境的抵抗能力 ,尤其是与病菌的侵入和感染关系非常密切[6] 。Ｐ是植物最重要的营养元素之一 ,大多数土壤都具有很强的固定Ｐ的能力 ,Ｐ肥的利…     

Changes of tomato rhizosphere microflora following application of the herbicide diphenamid to soil infested with Fusarium oxyspomm f.sp. lycopersici

Significant variations were detected in species composition between untreated rhizosphere and nonrhizosphere soils of tomato plants. Application of different concentrations of active ingredient of the herbicide diphenamid (5–250 ppm) to these soils caused significant alterations in species assemblages as compared with untreated soils. Also variations in species composition were denoted between treated rhizosphere and non-rhizosphere soils.Diphenamid concentrations of 10–100 ppm significantly affected microbial counts in soil and rhizosphere of tomato plants. Counts have been stimulated at diphenamid concentrations ranging from 10–50 ppm for fungi and 10–100 ppm for bacteria. At concentrations higher than the upper limits of these ranges, R/S values were not significantly affected.The results also indicated that Fusarium oxyspomm f.sp. lycopersici populations were unaffected by diphenamid at the recommended field rate (10 ppm). Above this concentration and within the conditions of the experiment, the pathogen maintained its population at detectable inocula. Population counts of Aspergillus candidus, a species reported to be able to degrade diphenamid, were high in both treated rhizosphere and non-rhizosphere soils.     

Ammonium Nutrition as a Strategy for Cadmium Mobilisation in the Rhizosphere of Sunflower

Ammonium nutrition of higher plants results in rhizosphere acidification due to proton excretion by root cells. The acidification induced by ammonium-fed plants can be exploited to promote a localised metal mobilisation in neutral to alkaline polluted soils and therefore to improve phytoextraction. The effects of ammonium uptake by sunflower (Helianthus annuus L.) plants on the external medium pH, aerial and root growth and tolerance to soluble Cd were studied in hydroponic culture. The ammonium-fed sunflowers induced a strong acidification of the solution and, compared to the nitrate-fed sunflowers, a small modification in mineral nutrition and a different Cd partitioning between root and shoot. Moreover, ammonium nutrition was found to induce a great mobilisation of a sparingly soluble form of cadmium (CdCO₃). A pot experiment studied the ability of different ammonium-based fertilisers (ammonium sulphate, ammonium thiosulphate, urea) to modify bulk and rhizo-soil pH, compared to the effect of calcium nitrate and to the unfertilised soil. Furthermore, in order to promote the persistence of ammonium in soil, a combined treatment of ammonium sulphate and DMPP, a nitrification inhibitor, was tested. Soil pH was strongly modified by chemical and biological processes involved in fertiliser transformations. In particular, due to nitrification, all ammonium-based treatments showed a bulk soil acidification of over 1.5 pH units and a relative increase in rhizo-soil pH as a consequence of nitrate uptake. The treatment with DMPP showed an opposite trend with a lower pH in rhizo-soil than in bulk soil. The ability of ammonium-fed plants to mobilise heavy metals from the non-labile pool was studied in another pot experiment using three soils with different properties and at different degree and type of heavy metal contamination. Whatever the soil, the metal concentrations in shoots were higher in plants fed with ammonium (ammonium sulphate plus DMPP treatment). Our results support the hypothesis that ammonium nutrition with nitrification inhibitors is a viable strategy to improve heavy metals phytoextraction while protecting bulk soil from acidification and presumably from metal leaching. An erratum to this article is available at .     

花椒叶浸提液对土壤微生物数量和土壤酶活性的影响

通过用花椒叶浸提液浇灌盆栽花椒幼苗,研究浸提液对土壤酶和土壤微生物的影响.结果表明, 花椒叶浸提液使根际土中细菌、真菌和放线菌数量以及微生物总数均有不同程度的减少,根际土中真菌和放线菌的数量变化呈降-升-降-升的趋势.20、60和80 g·L^-1浓度的叶浸提液使非根际土中细菌的数量显著增加21.59%、107.55%和8.62%,而40 g·L-1浓度的叶浸提液则使非根际土中细菌数量显著降低22.56%.叶浸提液使根际土蛋白酶、蔗糖酶和酸性磷酸酶活性明显低于非根际土相应的酶活性,而过氧化氢酶和多酚氧化酶活性则显著升高.土壤的蛋白酶活性与蔗糖酶活性呈显著正相关,与土壤放线菌数量呈显著负相关;多酚氧化酶活性与蔗糖酶活性呈显著负相关,与细菌、真菌、放线菌以及微生物总数呈显著正相关;放线菌只与蛋白酶、多酚氧化酶、蔗糖酶3种酶活性及真菌呈显著相关,与过氧化氢酶、酸性磷酸酶以及细菌和微生物总数的相关性均不显著.     

Effect of Root Agglutinin on Microbial Activities in the Rhizosphere

A total of 220 bacterial isolates were obtained from pea rhizosphere and nonrhizosphere samples. Of these samples, 100 isolates were chosen randomly to test for their agglutinative reaction against pea root exudate. The percentage of positive agglutination of bacteria isolated from the nonrhizosphere sample was significantly lower than that of bacteria isolated from the rhizosphere sample. Moreover, this agglutinative reaction could not be blocked either by treating the bacterial cells or root exudate with different carbohydrates before they were mixed or by boiling the root exudate first. Bacteria that could be agglutinated by pea root exudate followed the downward growth of the pea root through the soil profile. The greater abilities of such bacteria to colonize the pea rhizosphere were indicated by their higher rhizosphere-colonizing (rhizosphere/nonrhizosphere) ratios, whether the bacteria were added alone or together with nonagglutinating bacteria. However, bacteria did show different agglutinative reactions toward root exudates obtained from different plants.     

Bioengineering of a phytoremediation plant by means of somatic hybridization

Phytoremediation is a technology that exploits a plant's ability to remove contaminants from the environment or render toxic compounds harmless. An efficient metal phytoremediating plant must combine high biomass production and established cultivation methods with high tolerance to a specific contaminant and ability for root uptake, translocation, and compartmentalization of contaminants in the above-ground biomass. Symmetric and asymmetric somatic hybridizations were used to introduce toxic metal-resistant traits from Thlaspi caerulescens into Brassica juncea. B. juncea hypocotyl protoplasts were fused with T. caerulescens mesophyll protoplasts. The hypocotyl protoplasts of B. juncea were stained with CFDA before fusion and thus fluoresced green under UV, whereas the mesophyll protoplasts of T. caerulescens had red autofluorescense. Heteroplasmic fusion products were identified and selected by flow cytometry and cell sorting. All putative hybrids grown in the greenhouse had morphological characteristics of B. juncea. A Thlaspi-specific repetitive sequence was hybridized to total DNA of plants, including the parental species. All plants from both symmetric and asymmetric fusions showed Thlaspi-specific hybridization patterns while B. juncea did not exhibit any hybridization signal. Hybrid plants, produced by asymmetric somatic hybridization between the two species, demonstrated high metal accumulation potential, tolerance to toxic metals, and good biomass production.     

Enhancement of Population Densities of Pseudomonas putida PpG7 in Agricultural Ecosystems by Selective Feeding with the Carbon Source Salicylate

Sodium salicylate (1,000 μg/ml) was delivered through a drip irrigation system to agricultural field soils planted to tomato and infested with Pseudomonas putida PpG7, the host of the salicylate catabolic plasmid NAH7. In nonfumigated soils infested with approximately 10³ CFU of PpG7 per g in the top 30 cm, population densities were increased up to 112-fold within 14 days of the initial application of salicylate compared with the densities in the respective nonamended soils. Mean season-long population densities of PpG7 in the top 30 cm of soil were significantly increased (P < 0.01) from 216 CFU/g in nonamended soils to 1,370 CFU/g in salicylate-amended soils. In the respective rhizosphere soils, mean population densities of PpG7 were significantly increased (P < 0.01) from 92 to 2,066 CFU/cm of root. Soil fumigation interacted (P < 0.01) with salicylate amendment and further increased the mean population densities of PpG7 in nonrhizosphere soil by an additional 5,689 CFU/g of soil. This fumigation effect was not detected in rhizosphere soils. The effect of salicylate in increasing population densities of PpG7 in soil also was affected by inoculum level, field site, and soil depth. Proportionate differences were greater in soils infested with approximately 10³ CFU of PpG7 per g than in comparable soils infested with 10⁵ CFU/g. In low-inoculum soils, increases from salicylate amendments were 26- and 29-fold in rhizosphere and nonrhizosphere soils, respectively, and in high-inoculum soils, the respective increases were 5.6- and 5-fold. No increases of fungi able to utilize salicylate were detected in soils amended with salicylate. However, soil fumigation with metham-sodium significantly reduced (P < 0.01) population densities of fungal salicylate utilizers in rhizosphere and nonrhizosphere soils.     

Metabolic Status of Bacteria and Fungi in the Rhizosphere of Ponderosa Pine Seedlings

We determined the quantity and metabolic status of bacteria and fungi in rhizosphere and nonrhizosphere soil from microcosms containing ponderosa pine seedlings. Rhizosphere soil was sampled adjacent to coarse, fine, or young roots. The biovolume and metabolic status of bacterial and fungal cells was determined microscopically and converted to total and active biomass values. Cells were considered active if they possessed the ability to reduce the artificial electron acceptor 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) to visible intracellular deposits of INT formazan. A colorimetric assay of INT formazan production was also used to assess dehydrogenase activity. INT-active microorganisms made up 44 to 55% of the microbial biomass in the soils studied. The proportion of fungal biomass that exhibited INT-reducing activity (40 to 50%) was higher than previous estimates of the active proportion of soil fungi determined by using fluorescein diacetate. Comparison between soils from different root zones revealed that the highest total and INT-active fungal biomass was adjacent to fine mycorrhizal roots, whereas the highest total and active bacterial biomass was adjacent to the young growing root tips. These observations suggest that fungi are enhanced adjacent to the fine roots compared with the nonrhizosphere soil, whereas bacteria are more responsive than fungi to labile carbon inputs in the young root zone. Colorimetric dehydrogenase assays detected gross differences between bulk and rhizosphere soil activity but were unable to detect more subtle differences due to root types. Determination of total and INT-active biomass has increased our understanding of the role of spatial compartmentalization of bacteria and fungi in rhizosphere carbon flow.     

Rhizosphere bacterial populations of metallophyte plants in heavy metal-contaminated soils from mining areas in semiarid climate

Rhizosphere bacterial populations associated with four metallophyte plants in one of major polymetallic (Pb–Zn–Cu) semiarid Moroccan Hercynian province (Draâ Sfar, Marrakech, Morocco) presenting long-term contamination mainly with Zn and Pb were analysed and compared to selected control soils. In the highly Zn-, Cu-, Pb- and Cd- contaminated soils, the total number of culturable heterotrophic bacteria were found in low proportions (< 2.6 × 10² – 1.6 × 10⁴ g^–1soil). This bacterial content was slightly similar to that found in moderately polluted and controls soils (6.7 × 10⁴ – 5.8 × 10⁶). However, the bacterial diversity and the rhizosphere/soil ratio, which compares the bacterial content (or bacterial charge) around the metallophyte plants with that in non-rhizosphere soil, were the bacteriological parameters mostly affected by heavy metal contamination. The chronic Zinc-stress results in an increase of tolerance to this metal of both the rhizosphere and non-rhizosphere bacterial communities. However, in general, the rhizosphere bacterial populations exhibited less tolerance to Zn toxicity than the bacterial population of non-rhizosphere soils. This result suggests that toxic effects of Zn decrease in the rhizosphere soils of the metallophyte plants.     

番茄根际微生物种群动态变化及多样性

采用盆栽试验的方法对番茄根际主要微生物种群在不同生育期的动态变化进行了跟踪研究.结果表明,在番茄整个生育期内,可培养细菌数量在初花期和初果期时最多;放线菌数量从苗期到末期逐渐减少;真菌数量逐渐增多.番茄对细菌根际效应明显.DGGE图谱显示不同生育期番茄根际均具有较高的细菌多样性.根际细菌种类和数量在初花期发生较为显著的变化,初果期根际群落多样性指数(H)和物种丰度(S)值都达到最高,微生物最丰富,是筛选拮抗菌的较好时期.     

菌根真菌磷酸酶活性对红三叶草生境中土壤有机磷亏缺的影响

利用PVC分室培养装置研究了菌根际和菌丝际磷酸酶活性变化与土壤有机磷亏缺间的关系,结果表明,施用有机磷（植酸钠）能促进菌根根系侵染、提高土壤磷酸酶尤其是酸性磷酸酶的活性,使菌丝际范围变宽。菌丝际的存在使土壤有机磷亏缺范围加大,与非菌根植物相比,由于菌根真菌的作用,植物能更容易地从有机磷中获得磷营养以满足植物生长的需要,从而使其干物重和磷吸收量更高。