• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    An agent which ameliorates apparent toxicity of one or more heavy metals to a plant.
    公开号:CA2262245A1
    申请号:C2262245
    申请日:1999-02-18
    公开日:1999-08-18
    发明作者:Bernard R. Glick
    申请人:Bernard R. Glick；
    IPC主号:C12N1-20
    专利说明:
    [1" class="description-paragraph] METHOD AND BACTERIUM TO REDUCE HEAVY METAL TOXICITY IN PLANTS FIELD OF INVENTION The present invention is related to the field of soil remediation and in particular, phytoremediation of metals. More particularly, the present invention is concerned with the enhancement of plant vitality and capacity for bioremediation in soil containing high concentrations of heavy metals such as nickel, lead and zinc through association with phytosiderophore producing bacteria. BACKGROUND Pollution of the biosphere by toxic metals has accelerated dramatically since the beginning of the industrial revolution. Primary sources of this pollution include burning of fossils fuels, mining and smelting of metalliferous ores, municipal wastes, fertilizers, pesticides, and sewage. Toxic metal contamination of ground water and soil poses a major environmental and human health problem and is currently in need of an effective and affordable technological solution.Moreover, unlike organic pollutants, metals cannot be degraded to harmless products, such as carbon dioxide, but rather persist indefinitely in the environment, complicating their remediation. Living plants have the ability to accumulate heavy metals from soil and water, in particular those heavy metals which are essential for their growth and development (Mehra and Farago 1994). Certain plants also have the ability to accumulate heavy metals which have no known biological function (Bollard 1983). However, excessive accumulation of these metals can be toxic to most plants. Heavy metals ions when present at an elevated level in the environment, are adsorbed by roots and translocated to different plant parts, thereby leading to impaired metabolism and reduced growth (Foy et al. 1978; Bingham et al. 1986). Phytoremediation, the use of green plants to remove, contain, or render harmless environmental contaminants is considered to be an attractive alternative to the approaches that are currently in use for dealing with heavy metal contamination (Cunnigham and Ow 1996; Cunnigham and Berti 1993; Cunnigham et al. 1995; Salt et al. 1995). Phytoremediation of metals might take one of several forms: phytoextraction, rhizofiltration or phytostabilization. Phytoextraction refers to processes in which plants are used to concentrate metals from the soil into roots and shoots of plants; rhizofiltration is the use of plant roots to remove metals from effluents; and phytostabilization is the use of plants to reduce the mobility of heavy metals (and thereby reduce the spread of these metals in the environment). Recently, metal-tolerant plants have been used to vegetate and control soil erosion on metal mine tailings and waste piles, i.e., phytostabilization (Salt et al.1995; Cunningham and Lee 1995). Moreover, there are a number of reports of using metal accumulating plants to remove toxic metals from soil, i.e., phytoextraction (also called phytodecontamination) (Cunnigham and Ow 1996; Cunnigham and Berti 1993; Cunnigham et al. 1995; Kumar et al. 1995; Baker et al. 1991 ). However, it is believed that the accumulation of metal imposes a stress
    [2" class="description-paragraph] -2-on the plants and it is well documented that plants respond to a variety of different environmental stresses by synthesizing "stress" ethylene (Abeles et al. 1992;Hyodo 1991 ). In turn, the ethylene can trigger a stress/senescence response in the plant which may lead to the death of those cells where the ethylene is produced. One way to lessen the deleterious effects of heavy metals taken up from the environment on some plants has been through the use of soil bacteria or mycorrhizal fungi. It has been shown that the presence of ectomycorrhizal or vesicular-arbuscular (VAM) fungi on the roots of plants decreased the uptake of metals by the plant and thereby increased plant biomass (Tam 1995; Bradly et al.1982; Brown and Wilkins 1985; Heggo et al. 1990; Dueck et al. 1986; Killham and Firestone 1983). Similarly, chromium resistant pseudomands, isolated from paint industry effluents, were able to stimulate seed germination and growth of Triticum aestivus in the presence of potassium bichromate (Hasnain and Sabri 1996). In this case, the bacterial enhancement of the seedling growth was associated with reduced chromium uptake. Thus, while in both cases of soil bacteria or ectomycorrhizal fungi there was enhanced life of the plants it was done so with less heavy metal taken up overall. Consequently, what is needed is a means for enhancing the vitality of these plants growing in soil contaminated with heavy metals so that they are able to accumulate more heavy metals.
    [3" class="description-paragraph] -3-SUMMARY OF INVENTION In the natural environment, the roots of plants interact with a large number of different microorganisms and the nature of these interactions together with soil conditions are major determinants of the extent to which various plants grow and proliferate (Lynch 1990; click 1995). It has been previously reported that many plant growth promoting bacteria, free-living soil bacteria that are involved in a beneficial association with plants, contain the enzyme ACC deaminase (click 1995; Jacobson et al. 1994; click et al 1995). It was hypothesized that this enzyme, which has no known function in bacteria, might be part of a, hitherto undescribed, mechanism that certain bacteria use to stimulate plant growth. This could occur by ACC deaminase modulating the level of ethylene in developing plants (click et al 1994; Hall et al 1996; and click et al. 1997). A significant portion of the damage to plants infected with fungal phytopathogens occurs as a direct result of the response of the plant to the increased levels of stress ethylene (Van Loon 1984). Not only does exogenous ethylene often increase the severity of a fungal infection; but, as well, inhibitors of ethylene synthesis can significantly decrease the severity of a fungal infection. Since the enzyme ACC deaminase, when present in plant growth promoting bacteria, can act to rnodulate the level of ethylene in a plant, we sought to test whether such bacteria might lower the stress placed on plants by the presence of heavy metals, and therefore ameliorate some of the apparent toxicity of heavy metals to plants. Thus, in its broad aspect the present invention provides an agent which ameliorates apparent toxicity of one or more heavy metals to a plant. In
    [4" class="description-paragraph] -4-particular, such an agent may be a bacterium. According to two preferred embodiments this agent may be K, ascorbata SUD 165 or it may be K.ascorbata SUD 165/26. S According to another aspect of the present invention there is provided an agent which ameliorates apparent toxicity of Ni++ to a plant, and this agent can be a bacterium. In particular it may be K. ascorbata SUD 165 or K. ascorbata SUD165/26. According to a further broad aspect of the present invention there is provided a siderophore producing, plant growth promoting bacteria which ameliorate apparent toxicity of one or more heavy metals to a plant. According to one embodiment the bacteria may be K. ascorbata SUD 165. According to another embodiment it may be K. ascorbata SUD 165/26. A heavy metal which is ameliorated according to this aspect is Ni++ According to a further broad aspect the present invention provides siderophore producing, plant growth promoting bacteria which ameliorate apparent toxicity of one or more heavy metals to a plant and reduce the amount of ethylene that evolves from such a plant. Preferred embodiments include bacteria known as K. ascorbata SUD 165 or K. ascorbata SUD 165/26. According to a further aspect there is provided siderophore producing, plant growth promoting bacteria which ameliorate apparent toxicity of Ni++ to a plant and reduce the amount of ethylene that evolves from such a plant. Preferred
    [5" class="description-paragraph] -5-embodiments include bacteria known as K. ascorbata SUD 165 or K. ascorbata SUD 165/26.As will be readily apparent to those skilled in the art, the present invention can be applied at any stage of plant life from seedling to fully mature plant, and may be applied to a wide variety of plants including those specifically disclosed herein, namely tomato and canola. The present invention also provides a method to ameliorate apparent toxicity of one or more heavy metals to a plant and reduce the amount of ethylene that evolves from said plant which consists of adding a sufficient amount of siderophore producing, plant growth promoting bacteria to such a plant. As is apparent, preferred embofiments of the method include the siderophore producing, plant growth promoting bacteria known as K. ascorbata SUD 165/26 or K. ascorbata SUD 165.As will also be readily apparent to those skilled in the art, the method of the present invention includes application of a growth promoting agent of the present invention at any stage of plant life from seedling to fully mature plant, and includes application to a wide variety of plants including those specifically disclosed herein, namely tomato and canola.
    [6" class="description-paragraph] -6-BRIEF DESCRIPTION OF THE FIGURES FIGURE 1. Growth of K. ascorbata SUD165 at different temperatures. FIGURE 2. The influence of Ni++ on the growth (as reflected by absorbence) of K.ascorbata SUD165. Cells were grown for 18 hours in TLPmedium plus gluconate at 25°C. FIGURE 3. Siderophore biosynthesis by K,ascorbata SUD165. The amount of siderophores secreted to the growth medium is presented as the desferal equivalents. FIGURE 4. The influence of [Ni++] on canola seed development in growth pouches. The error bars represent 1 S.E.M. Values differ significantly (P<0.001 ) from control group. Data were analyzed by ANOVA with 50-60 seedlings in each treatment group. The results of a typical experiment are shown. FIGURE 5. The influence of [Ni++] on canola seed development in pots. The error bars represent I S.E.M. Values differ significantly (P<0.001) from the control group. Data were analyzed by ANOVA with 55-60 seedlings in each treatment group. The results of a typical experiment are shown. FIGURE 6. The influence of [Ni++] on tomato seed development in pots. The error bars represent I S.E.M. Value differ significantly (P<0.001 ) from the control group. Data were analyzed by ANOVA with 55-60 seedlings in each treatment group. The results of a typical experiment are shown. FIGURE 7. Siderophore biosynthesis as a function of time in culture by K.ascorbata SUD165/26. The amount of siderophore produced in the growth medium is presented as the desferal equivalents. DETAILED DESCRIPTION OF THE INVENTION The invention will be further described with reference to the following examples. These examples are provided for illustrative purposes only and should not be used to limit the scope of the invention. Selection of nickel resistant bacterial strain. Nickel is an essential micronutrient for many microorganisms (Hausinger 1987). However, at millimolar concentrations nickel inhibits the growth of most wild type bacteria and is tolerated by only a minority of microorganisms (Schmidt and Schlegel 1989). Described herein is such a tolerant microorganism. We isolated nickel resistant bacteria of the present invention from highly polluted nickel- and copper-containing soil (Table 1 ) using a spread plate procedure on a pH-neutral TLP medium. This medium is designed to avoid precipitation of heavy metal salts at I mM concentration. In total there were approximately 4 x 103 nickel resistant bacteria per gram dry weight of soil, or about 1 % of the total bacterial population in the soil. In order to isolate the plant growth promoting bacteria of the present invention, all of the nickel resistant isolates were tested for the ability to grow on minimal medium with 1-aminocyclopropane-1-carboxylate (ACC) as the sole source of nitrogen (click et al. 1995). The method for isolating such strains has been described in U.S. application 08/551,244 which is incorporated herein by reference. Approximately 7% of the nickel tolerant strains also had the ACC +phenotype.Finally, the ability of nickel resistant strains that were able to grow on ACCto produce siderophores was tested. The best siderophore producing strain (designated SUD165) is an embodiment of the plant growth promoting bacteria of the present invention. Identification and properties of the SUD165 strain. The microorganism isolated is Gram-negative, motile, methyl red positive, indole positive, and citrate positive. On MacConkey agar it fermented D-glucose, _g_ D-galactose, D-mannitol, D-mannose, D-sorbitol, L-arabinose, and sucrose, but not lactose. SUD165 cells grew on M9 minimal medium with above mentioned sugars and salicin. The bacterium did not grow on M9 with lactose. On nutrient agar or solid TLP medium plus gluconate, the bacterium formed circular white colonies with an "entire" edge. On solid TLP medium plus gluconate, the bacterial colonies had a metallic-blue sheen. These results suggest that the bacterium belongs to the family Enterobacteriaceae. The SUD165 strain was subsequently characterized by fatty acid analysis by Microbial ID, Inc. (Newark, DE) as Kluyvera ascorbata (Farmer et al. 1981 ). Kluyvera ascorbata SUD165 was able to grow in nutrient broth (Difco) at temperatures from 5°C to 37°C suggesting that the bacterium should be considered to be a psychrotroph (Fig. 1 ). On solid TLP medium with gluconate, the strain was found to be resistant to 1 mM Ni++, 1 mM Zn++, 1 mM Pb++, 0.1 mM Cr04-, 100 pg/mL penicillin G, 25 Ng/mL carbenicillin and 25 Ng/mL ampicillin.It was sensitive to 0.01 mM Hg++, 0.25 mM Co++, 0.25 mM Cd++, 0.25 mM Cu++as well as to 50 pg/mL streptomycin, 20 Ng/mL tetracycline, 50 Ng/mL kanamycin and 25 pg/mL chloroamphenicol. The effect of nickel on K. ascorbata SUD165,.growth. Ten mL of TLP medium containing 0.2% gluconate and varying concentrations of nickel chloride in 250 mL Erlenmeyer flasks was inoculated by the addition of 0.1 mL of an overnight culture of K. ascorbata SUD165 pregrown in the same medium without nickel. After 18 h of growth at 25°C in a rotary shaker (200 rpm) the absorbance at 600 nm of the cell suspensions were measured. At all of the concentrations of Ni++ tested cell growth was inhibited to some extent (Fig. 2). However, when added to non growing bacterial suspension in distilled water, the metal did not decrease the amount of colony forming units in the suspension after 5 days of incubation at 25°C (data not shown). Siderophore Production by K. ascorbata SUD165 Siderophore production by K. ascorbata that was grown in King's medium Bwithout phosphate (Raaska et al. 1993) was measured (Fig. 3). A peak of siderophore production was detected following approximately 44 h of incubation. After that the amount of siderophore present in the growth medium decreased. Plant growth promoting activity of K. ascorbata SUD165. Since K. ascorbata was selected on the basis of its ability to utilize ACC as a sole source of nitrogen, it was reasonable to hypothesize that it might contain ACC deaminase activity and that it might be able to stimulate the growth of plant roots by hydrolyzing ACC from germinating seeds thereby lowering the level of ACC, and hence the level of ethylene in seeds. In fact, a cell free extract of K.ascorbata SUD165 cells displayed a low level of ACC deaminase activity; 26 nmoles/mg/h as compared to 300 nmoles/mg/h for the well characterized plant growth promoting bacterium Pseudomonas putida GR12-2 (click et al 1994 and 1997). Indeed, K. ascorbata SUD165 stimulated canola root elongation only when the light level was was very low (Table 2). K. ascorbata SUD165 cells have the ability to bind to canola seed coats as visualized by scanning electron microscopy (not shown). Effect of nickel on canola and tomato seedlingis The results of a series of experiments to consider the sensitivity of canola to Ni++ cations in growth pouches are shown in Fig. 4. As can be seen) canola seeds developed normally even in the presence of up to 0.1 mM nickel chloride. Above this concentration, plant root and shoot elongation is inhibited. In pot experiments, i.e., in the presence of soil, a higher concentration of Ni++ was required to noticeably inhibit canola root and shoot length than in growth pouch experiments (Fig. 5). The apparent lower level of toxicity of Ni++ in soil most probably represents the binding of Ni++ to soil particles thereby making the cation unavailable to the developing seedlings. Tomato plants grown in pots were somewhat more sensitive to Ni++ than canola plants grown in pots (Fig. 6). With both tomatoes and canola grown in pots, roots appeared to be more sensitive to the inhibitory effects of Ni++ than shoots. With canola seedlings in growth pouches, roots and shoots were equally sensitive to growth inhibition by Ni++.Effect of K. ascorbafa SUD165 on toxicity of nickel to canola and tomato seedlings The effect of adding K. ascorbata SUD165 to canola or tomato seeds prior to germinating the seeds, either in a growth pouch (canola only) or in pots (canola and tomato), in the presence of inhibitory amounts of Ni++ is shown in tabular form (Tables 3-5). The results are presented as a tolerance index, making it easier to compare the effects of different treatments. A tolerance index of 1.0 indicates that the treatment was not inhibitory, while a tolerance index of 0.1 indicates that the growth of treated plants was only 10% of the growth of the control. The data presented in Tables 3-5 shows that at all levels of nickel tested (1-6 mM Ni++), using both a low and a high bacterial cell treatment (cell suspension absorbance of 0.025 or 0.50), with both canola and tomato plants, with both roots and shoots, and both in gnotobiotic growth pouches and in pots, the addition of K. ascorbata SUD165 significantly decreased the toxicity of the added nickel. The effect was highly reproducible in spite of variation in the root and shoot lengths. Moreover, the "protective" effect of K. ascorbata SUD165 increased as the density of the cell suspension increased.6sNi++ accumulation by canola seedlings Since plants growing in metal-enriched environments take up nickel to varying degrees in response to external and internal factors (Reeves 1988), it is important to assess whether the addition of K. ascorbata SUD165 affects the uptake of nickel by canola seedlings. As can be seen in Table 6, the presence of K. ascorbata SUD165 did not change the amount of nickel taken up per mg dry weight of either roots or shoots. By this measure, although K. ascorbata decreased the toxicity of Ni++ to canola, it had no influence on amount of Ni++accumulated by the plant. Ethylene production by plants grown in the presence of nickel When a suspension of K. ascorbata SUD165 cells was added to canola seedlings grown in gnotobiotic growth pouches in the presence of 2 mM Ni++ the amount of ethylene that was evolved over a period of 18 h decreased from 590 t 182 nmoles/mg dry weight in the absence of the bacterium to 275 t 90 nmoles/mg dry weight in the presence of the bacterium. The variation notwithstanding, ethylene levels were always higher in the absence of the bacterium each of the four times that this measurement was performed. Properties of siderophore hyperproducing mutant K, ascorbata SUD165/26 Colonies of 8-hydroxyquinoline resistant mutants of K. ascorbata SUD165 of the present invention were suspended in 1 ml of saline and then 5 ,ul of the suspension were applied to blue agar plates containing the ternary complex chrome azurol S/iron(III)/hexadecyltrimethylammonium bromide which served as an indicator (Schwyn and Neilands 1987). Two of the 28 mutants which developed larger (in comparison with the wild type) orange haloes around the colonies were chosen for quantitative assay of siderophore(s) accumulation in liquid culture. These two strains showed a dramatic increase in siderophore(s) production compared to the wild type. One of the them, namely K. ascorbata SUD165/26) was used as outlined below. A sample of K.ascorbata SUD165/26 has been deposited at the International Depositary ATCC on November 6, 1997 bearing accession number ATCC 202052. Just like wild type strain K.ascorbata SUD165/26 cells were resistant to Ni++, Zn++, Pb++ and Cr04-. The mutant strain demonstrated ACC deaminase activity (31.4 nmoles/mg protein/h) that was similar to the level of activity found in the wild type bacterium (26 nmoles/mg protein/h). Strain K. ascorbata SUD165/26 produced 50-150 times more siderophore then the wild type, and a peak of siderophore biosynthesis following approximately 3 days of cultivation in phosphate free King's medium B at 30°C(see Fig. 7). Effect of K.ascorbata SUD165 and SUD165/26 on nickel toxicity to tomato seedlings in a long term experiment Nickel at low concentrations has been reported to be a plant growth stimulant (Mishra and Kar 1974). In agreement with this report, tomato seeds sown in pots irrigated with 1 mM nickel chloride produced more wet weight than with pots irrigated with water. On the other hand, treatment of plants with nickel significantly decreased the plant dry weight. After approximately four weeks of cultivation, nickel treated plants started thinning at the base of the stem and eventually fell down. By contrast, in the presence of K. ascorbata 165 of: K. ascorbata 165/26 nickel treated tomato plants behaved similarly to plants with no nickel, i.e., there was no apparent thinning of the stem and the plants did not fall down. The data shown in Table 7 indicates that K. ascorbata SUD~65 and K.ascorbata 165/26 significantly increased stem length, and dry- and wet-weights of tomato seedlings in both the absence and the presence of nickel. The data also revealed that in the presence of nickel the protein content in plant leaves decreased. The K.ascorbata SUD165 strain, and to an even greater extent the K.ascorbata SUD165/26 strain alleviated the toxic action of nickel by increasing the protein level in tomato seedlings.Thus) the K. ascorbata SUD165 of the present invention is highly effective at protecting short term growth of plants from growth inhibition caused by the presence of high concentrations of nickel. However, on a dry weight basis, the plant grown in the presence and absence of the bacterium took up approximately the same amount of nickel so that it is unlikely that the bacterium is somehow limiting nickel uptake by the plant. A likely explanation of the data, although, by no means is the invention limited by the proposed explanation, is that, in the plants in the short term growth examples above, the bacterium protects the plant against the inhibitory effects of nickel-induced stress ethylene formulation. In this regard, (i) heavy metal can induce ethylene production by plants (Weckx et al. 1993), (ii) an excess of ethylene can inhibit plant development (Jackson 1991 ), and (iii) the direct promotion of plant root growth by a number of different soil bacteria is based on the ability of bacterial ACC deaminase to hydrolyze and decrease the amount of ACC, an ethylene precursor, in plants and, as a result, to decrease ethylene biosynthesis by plants (click et al 1994; Hall et al 1996; click et al.1997). Moreover, with canola seedlings grown in the presence of high levels of nickel it was observed that the addition of the bacterium K. ascorbata SUD165 caused a significant decrease in ethylene production. Another possible explanation of the present invention, although by no means is the invention limited by the following proposed explanation, is related to siderophore production by K. ascorbata SUD165. In this regared at least in part, the toxic effect of nickel appears to be due to an induced iron deficiency. The siderophore producing bacteria of the present invention may reduce nickel toxicity by supplying the plant with iron. In fact, the results obtained in the longer term examples support this possibility. In the presence of added bacteria, the chlorophyll concentration in nickel treated plants increased significantly (see Table
    [7" class="description-paragraph] 7). Moreover, the greatest elevation of chlorophyll content in plant leaves in the presence of nickel was promoted by the siderophore(s) hyperproducing mutant K.ascorbata SUD965/26. (Table 7) The addition of the bacterium did not influence the nickel level in shoots or roots of the plant. These results are in agreement with the results reported in Table 6, namely that nickel levels in 5-7 day old plant seedlings are independent of the presence of an added bacterium. The siderophore hyperproducing mutant K.ascorbata 165/26 protected tomatoes against nickel toxicity to a greater extent than the wild type K.ascorbata SUD 7 65. Thus, at least part of the toxic effects of some heavy metals, including nickel, in plants, results from an induced iron deficiency, and there is evidence that increasing the supply of iron can reduce the severity of nickel toxicity (Bollard 1983; Foy et al. 1978; Bingham et al. 1986; Yang et al. 1996). Moreover, since bacterial siderphores can provide iron to various plants (Reid et al. 1986; Wang et al. 1993; Bar-Ness et al. 1991 ), restating what is only a hypothesis of the mechanism of action of the present invention, siderophores produced by K.ascorbata SUD165 may enhance the plant vitality and reduce nickel toxicity by supplying the plant with iron thereby eliminating iron deficiency. As alternative hypotheses the increased metal tolerance in plants as induced by the bacterium of the present invention might be related to the production of metal-binding compounds (metallothioneins); alteration of metal compartmentation patterns;modification of cellular metabolism; or changes in membrane structure (Turner 1994). Regardless of the precise mechanism used by the bacterium of the present invention it will be apparent to a person skilled in the art that production of bacteria capable of producing even higher concentrations of siderophore may be possible and all such bacteria are within the scope of the present invention.It will also be apparent to a person skilled in the art that any microorganism whether natural or artificially produced which is capable of producing concentrations of siderophore which are effective in enhancing the vitality of a plant growing in soil contaminated with heavy metals is within the scope of the present invention. Regardless of the precise mechanism used by the bacterium of the present invention to protect plants, the examples with plant seedlings described make it apparent to those skilled in the art that bacteria of the present invention and similar bacteria will find use in the development of phytoremediation strategies.In this regard, it is readily apparent that the present invention is not limited to nickel, but includes all heavy metals which may be removed from polluted soil either by increasing the metal accumulating ability of plants or by increasing the amount of plant biomass. It will also be apparent to a person skilled in the art that in heavily contaminated soil where the metal content exceeds the limit of plant tolerance, it may be possible to treat plants with plant growth promoting bacteria of the present invention, increasing plant biomass thereby stabilizing, revegetating and remediating metal polluted soils. Other uses apparent to those skilled in the art will readily come to mind and as such, while the present invention has been described with reference to illustrative embodiments, this description is not intended to be construed in a limiting sense. Materials and Methods Media The basic mineral medium used for isolation and growth of nickel resistant bacteria was the Tris buffered low-phosphate (TLP) medium described by Mergeay et al. (1985) supplemented by trace metals 1.0 mL of a stock solution of trace metals per L where the stock solution consists of, in g/L, FeS04~7H20, 0.2; ZNS04~7H20, 0.01; MNC12~4H20, 0.003; CoCI2~GH20, 0.02; CuCI2~GHz0, 0.001; NiCIZ~GH20, Na2Mo04~2H20, 0.5; H3B03, 0.031 (Bogardt and Hemmingsey 1992) and by 0.2% sodium gluconate. For solid medium, Difco Bacto-agar was added at a concentration of 2% (w/v). Stock solutions of nickel chloride (1 M and 0.1 M) were autoclaved and added to the medium as required. Cell counts were determined using nutrient broth (Difco). Utilization of carbon sources was investigated by supplementing M9 minimal medium (Miller 1972) with various organic compounds (0.2%; w/v). For testing resistance to antibiotics and metals, stock solutions were filter-sterilized and then added to sterile TLP medium. Selection of nickel resistant bacteria Soil samples were taken from metal-impacted wetlands near Sudbury, Ontario, an area which had been exposed to heavy metal contamination from mine wastes following the discovery of a massive nickel-copper ore body (Freedman and Hutchison 1980). The soil sample from which the selected nickel resistant plant growth promoting bacterium was isolated contained a range of different metals including high levels of both nickel and copper (Table 1 ). Twenty mL of TLP medium were added to two g of soil and the suspension as incubated at 25°C for two hours in a rotar shaker (400 rpm). The suspension was then allowed to stand for about one hour before 0.3 mL of supernatant was spread over solid TLP medium containin 0.2% (w/v) gluconate, trace elements and 1 mM NiCl2. Plates were incubated for two days at 30°C. Nickel resistant colonies were purified on the same medium and then tested for the ability to grow on TLP medium with 1-aminocyclopropane-1-carboxylic acid (ACC) as the only source of nitrogen. Siderophore assax Following growth on King's medium B without phosphate (Raaska et a1.1993), siderophores secreted to the growth medium were detected and quantified according to the "universal" method of Schwyn and Neilands (1987).The siderophore desferal mesylate (Sigma) was used to produce a standard curve of from 0.5 to 25 mM siderophore. The incubation period for the standard was hours at room temperature. Root and shoot elongation assax Canola (8rassica campestris cv. Tobin) seeds were kindly provided by Dr. G. Brown (Agrium, Inc., Saskatoon., Sask.). Seeds were surface sterilized by soaking for 10 min in 1.5% sodium hypochlorite and then thoroughly rinsed with sterile distilled water. The sterilized seeds were incubated for 1 h at room temperature either in sterile distilled water as a blank control, or in a bacterial suspension in distilled water adjusted to an absorbance of 0.025 or 0.5 at 600 nm. Six canola seeds were placed in each seed-pack growth pouch (125 x 157mm;Mega International, MN) that had been filled with 10 mL distilled water or 10 mLNiCl2 and autoclaved 30 min at 121 °C prior the addition of the cells. Ten replicate pouches were used for each treatment. The pouches were incubated upright in a plastic tray partially filed amount of water sufficient to cover the bottom. The tray was covered with transparent Sarah Wrap T"". The pouches were incubated at 25°C for four days in a growth chamber with 12h photoperiod and a light intensity of 12.9 pmol/mZ/sec. The growth promoting effect of the bacterium was determined at a light intensity of 0.92 pmol/m2/sec. At the end of incubation the pouches were opened and the seedling root and shoot length were measured.The results of these experiments are presented as the tolerance index, TI (Wilkins 1978) where: TI = RLm/RL~ where RLm is the root length of plants grown in the presence of a specific added metal and RLc is the root length of plants grown in the absence of that metal. The tolerance index may also be expressed as the ratio of the shoot lengths of plants grown in the presence and absence of a specific added metal. Pot experiments (Tables 4 and 5) Plants were sown in plastic pots (77 mm top diameter, 55 mm bottom diameter, 66 mm high) filled with approximately 100 g air dried general purpose growth medium Pro-Mix (B/x) (General Horticulture, Inc., Red Hill, PA). Plants were grown in a growth chamber at 25°C (canola) or at 20°C (tomato) under 12h light/dark photoperiod with a light intensity of 12.9 pmol/m2/sec). The pots were irrigated with either 250 mL distilled water or a solution of nickel chloride and incubated four days for canola or ten days for tomato (Lycopersicon esculentum Mill. c.v. Heinz 1439VF 402A; Stokes Seeds Ltd., St. Catharines, Ontario). The moisture content of soil was maintained by placing each pot in a polyethylene bag. Thirty seeds were sown per pot, and 60 seeds were used per treatment. ACC deaminase determination ACC deaminase activity was determined by estimating (a-ketobutyrate production (nmoles/mg protein/h) according to the procedure of Honma and Shinomura (1978). NiCIZ u-ptake Seeds were sown in small pouches (60 x 40 mm) in the presence of 1 mL of ImM NiCl2 and 5 mCi/mL 63NiC12 (Amersham). Following four days of incubation at 25°C, shoots and roots were harvested separately and washed extensively, first in several changes of 0.01 M EDTA and then in distilled water to remove any nonspecifically bound radioactivity, dried overnight at 105°C and weighed. The amount of 63Ni++ accumulated by a plant root or shoot was determined using a liquid scintillation counter (Beckman LS1701 ). Each experimental point included three pouches with six seeds in each pouch. Cell suspension preparation For seed innoculation, cells were grown in TLP with 0.2% gluconate, 0.01 casamino acids, trace elements and 0.1 mM NiCl2 at 25°C until late log phase. Cells were then pelleted by centrifugation at 25000 x g for 10 min and washed twice with sterile distilled water. Bacterial suspensions in distilled water were adjusted to an absorbance at 600 nm of either 0.5 (equivalent to approximately 7.4 x 108 cfu-mL~') or 0.025 (i.e., 3.0 x 10' cfu-mL-') and then used for seed inoculation. Ethylene measurement The production of ethylene by canola seedlings was measured using a GOW-MAC model 69-750 gas chromatograph. Eight seeds were sown in each growth pouch (65 x 55 mm) containing 1 mL of 2 mM nickel chloride. Some seeds were pretreated with a bacterial suspension with an absorbance of 0.5 at 600 nm.Following incubation for four days in a growth chamber at 25°C under a 12h light/dark photoperiod with a light intensity of 12.9 Nmol/m2/sec, growth pouches were placed in sealed 250 mL bottles for 18 h before samples were taken for gas chromatographic analysis. Isolation of siderophore hyperaaroducing mutant The siderophore hyperproducing strain K. ascorbata SUD165/26 was selected from a population of wild type K. ascorbata SUD165 cells which were grown on solid Tris buffered low-phosphate medium (Mergeau et al. 1985) supplemented by trace elements (Bogard and Hemmingensen 1992) containing 0.2 mM 8-hydroxyquinoline. About 10' K. ascorbata SUD165 cells were placed on the agar plate and incubated at 30°C for 8 days at which point spontaneous 8-hydroxyquinoline resistant mutants were selected. The mutation frequency was about 10-6. Mutants were purified on the same solid medium. Long term pot experiment (Table For long term experiments tomato seeds (Lycopersicon esculentum Mill. c.v.Heinz 1439 VF, Stokes Seeds Ltd., St. Catherine, Ontario) were surface sterilized and incubated for 1 h either in a bacterial suspension with an absorbance adjusted to 0.5 at 600 nm in distilled water. The seeds were then sown in pots (155 mm top diameter, 104 mm bottom diameter and 145 mm high) filled with approximately 500 g wet weight (170 g dry weight) general purpose growth medium Pro-Mix 'BX' (General Horticulture, Inc., Red Hill, PA) containing, according to the manufacturer, sphagnum peat moss (75-85% by volume), perlite, vermiculite, macronutrients (calcium, magnesium, nitrogen, phosphorus, potassium, sulfur), mironutrients (boron, copper, iron, manganese, molybdenum, zinc), dolomitic limestone, calcitic limestone and wetting agent, The soil pH was approximately 6Ø Plants were grown at room temperature (20-22°C) under a 12h light/dark period with a light intensity of 32 pmol/m2/sec. Pots were irrigated with 100 ml ImM NiCl2 at days 0, 6, 12, 18 and 24. Tap water (100 ml) was added to the nickel treated pots at days 3, 9, 15, 21, 27 and then once every three days until the end of the experiment. Pots without added nickel were irrigated with ml of tap water once every three days. Five seeds were sown per pot with four pots per treatment. To overcome possible nutrient limitation during plant growth, 50 ml of fertilizer (0.18% urea , 0.1 % KCI) was supplied to each pot once a week.After 6 weeks of cultivation the plants were harvested. Roots were separated from shoots and seedling height was measured before the shoot biomass was oven-dried at 105°C for 24 h. Nickel analxsis Plants were harvested and roots were washed extensively in several changes of a solution containing 5 mM Tris-HCI buffer (pH6.0) with 5mM EDTA and then in distilled water in order to remove nonspecifically bound nickel ions. Shoots and roots were oven-dried at 105°C for 24 h. Aliquots (5-15 mg) of dried material were placed into disposable borosilicate tubes (74 mm high, 10 mm diameter) and then ashed at 500°C for 18-20 h. The ash was dissolved in a 0.4 ml of 70% (w/w) HN03 and 30% (w/w) HZOZ (1:1 ). The ash solution was heated for 2 h at 70°C and then distilled water added to a final volume of 3 ml. The nickel concentration was determined using a graphite furnace atomic absorption spectrometer SpectrAA-600 (Varian). The instrument was zeroed with 1 % HN03 blanks. Calibration were performed in the range of the analysis. Samples in triplicate were taken from each plants with 13-15 plants for each treatment. To analyze nickel in soil, samples were taken from each of the amended pots, 20 mm top layer soil samples were collected. The samples were dried for 24 h at 105°C, ground in a porcelain mortar, deionized water was added (1 g soil to 5 ml water) and the mixture was shaken for 30 min at room temperature. Protein assay For protein estimation, leaves were ground in a porcelain mortar and then extracted with 0.05M K-Na phosphate buffer pH7.2 containing 40 pgiml phenylmethylsulfonyl fluoride and 0.1 % SDS. Debris was pelleted by centrifugation and the protein content of the supernatant estimated by the method of Bradford (1976) using bovine serum albumin as a standard. Samples in duplicate were taken from each plant with 13-15 plants per treatment. Chlorophyrll assay Chlorophyll from plant leaves was measured by the method of Hiscox and Israelstam (1979). Samples were taken in duplicate from each plant with 13-15 plants per treatment. Statistical analysis Pouch and pot experiments were set up in a randomized block design. Data were analyzed by means of analysis of variance (ANOVA), and treatment means were compared by Duncan's multiple range test. The mean differences of the treatment was found to be significant at a level of P<0.001. References Abeles, F.B., Morgan, P.W. and Saltveit, M.E., Jr. 1992. Regulation of ethylene production by internal, environmental and stress factors. In Ethylene in Plant Biology, 2nd Edition, pp. 56-119, Academic Press, San Diego.2. Baker, A.J.M., Reeves, R.D., and McGrath, S.P. 1991. In situ decontamination of heavy metal polluted soil using crops of metal-accumulating plants. A feasibility study. In In situ bioreclamation. Application and investigation for hydrocarbon and contaminated site remediation. Edited by R.E. Hinchee, and R.F. Olfenbuttel. Butterworth-Heineman, pp. 600-605.3. Baker, A.J.M., and Walker, P.L. 1990. Ecophysiology of metal uptake by tolerant plants. In Heavy metal tolerance in plants: evolutionary aspects. Edited by A.J.Shaw. CRC Press, Boca Raton, pp. 155-157.4. Bar-Ness, E., Chen, Y., Hadar, Y., Marschner, H. and Romheld, V.1991.Siderophores of Pseudomonas putida as an iron source for dicot and monocot plants. Plant Soil 130:231-241.5. Bingham, F.T., Pereyea, F.J., and Jarrell, W.M. 1986. Metal toxicity to agricultural crops. Metal Ions Biol. Syst. 20:119-156.6. Bogardt, A.H. and Hemmingsey, B.B. 1992. Enumeration of phenanthracene-degrading bacteria by an overlayer technique and its use in the evaluation of petroleum contaminated sites. Appl. Environ. Microbiol. 58: 2579-2582.7. Bollard) E.G. 1983. Involvement of unusual elements in plant growth and nutrition. In Inorganic plant nutrition. Edited by A. Lauchli, and R.L. Bielsky. Encyclopedia of plant physiology, vol. 15B, Springer-Verlag, Berlin. pp. 695-744.
    [8" class="description-paragraph] 8. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein-dye binding. Anal. Biochem. 72:248-258.
    [9" class="description-paragraph] 9. Bradley, R., Burt, A.J., and Read, D.J. 1982. The biology of mycorrhyza in the Ericaceae. VIII. The role of mycorrhyzal infection in heavy metal resistance. New Phytol. 91:197-202.
    [10" class="description-paragraph] 10. Brown, M.T., and Wilkins, D.A. 1985. Zinc tolerance of mycorrhyzal Betula. New Phytol. 99:101-106.
    [11" class="description-paragraph] 11. Cunningham, S.D., and Berti, W.R. 1993. Remediation of contaminated soils withgreen plants: an overview. In vitro Cell. Dev. Biol. 29P:207-212.
    [12" class="description-paragraph] 12. Cunningham, S.D., Berti, W.R., and Huang, 1995. Phytoremediation of contaminated soils. Trends Biotechnol. 13:393-397.
    [13" class="description-paragraph] 13. Cunningham, S.D., and Lee, C.R. 1995. Phytoremediation: plant-based remediation of contaminated soils and sediments. In Bioremediation: science and application. Soil. Sci. Soc. Am. Special Publication 43:145-155.
    [14" class="description-paragraph] 14. Cunningham, S.D., and Ow, D.W. 1996. Promises and prospects of phytoremediation. Plant Physiol. 110:715-719.
    [15" class="description-paragraph] 15. Dueck, T.A., Visser, P., Ernest, W.H.O., and Schat, H. 1986. Vesicular-arbuscular mycorrhyzae decrease zinc toxicity to grasses in zinc polluted soil. Soil Biol. Biochem. 18:331-333.
    [16" class="description-paragraph] 16. Farmer, J.J., Fanning, G.R., Huntley-Carter, G.P., Holmes, B., Hickman, F.W., Richard, C., and Brenner, D.J. 1981. Kluyvera, a new (redefined) genus in the family Enterobacteriaceae: identification of Kluyvera ascorbata sp. nov.and Kluyvera cryocrescent sp. nov. in clinical speciments. J. Clin. Microbiol.13:919-933.
    [17" class="description-paragraph] 17. Foy, C.D." Chaney, R.L., and White" M.C. 1978. The physiology of metal toxicity in plants. Ann. Rev. Plant Physiol. 29:511-566.
    [18" class="description-paragraph] 18. Freedman, B., and Hutchison,T.C. 1980. Pollutant inputs from the atmosphere and accumulations in soils and vegetation near a nickel-copper smelter at Sudbury, Ontario, Canada. Can. J. Bot. 58:108-132.
    [19" class="description-paragraph] 19. Click, B.R., Jacobson, C.B., Schwarze, M.M.K., and Pasternak, J.J. 1994. 1-Aminocyclopropane-1-carboxylic acid deaminase mutants of the plant growth promoting rhyzobacterium Pseudomonas putida GR 12-2 do not stimulate canola root elongation. Can. J. Microbiol. 40:911-915.
    [20" class="description-paragraph] 20. Click, B.R., Karaturovic, D.M., and Newell, P.C. 1995. A novel procedure for rapid isolation of plant growth promoting pseudomonads. Can. J. Microbiol.41:533-536.
    [21" class="description-paragraph] 21. Click, B. R. 1995. The enhancement of plant growth by free-living bacteria. Can. J. Microbiol. 41, 109-117.
    [22" class="description-paragraph] 22. Click) B.R., Liu) C., Ghosh, S. and Dumbroff, E.B. 1997. Early development of canola seedlings in the presence of the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2. Soil Biol. Biochem., 29:1233-1239.
    [23" class="description-paragraph] 23. Hall, J.A., Peirson, D., Ghosh, S. and Click, B.R. 1996. Root elongation in various agronomic crops by the plant growth promoting rhizobacterium Pseudomonas putida GR12-2. Isr. J. Plant Sci. 44, 37-42.
    [24" class="description-paragraph] 24. Hasnain, S., and Sabri, A.N. 1996. Growth stimulation of Triticum aestivum seedlings under Cr- stresses by non rhizospheric pseudomonad strains. Abstracts: 7th International Symposium on Biological Nitrogen Fixation with Non-Legumes. Faisalabad) Pakistan. p.36.
    [25" class="description-paragraph] 25. Hausinger, R.P. 1987. Nickel utilization by microorganisms. Microbiol. Revs.3 5 51: 22-42.
    [26" class="description-paragraph] 26. Heggo, A., Angle, J.S., and Chaney, R.L. 1990. Effect of vesicular-arbuscular mycorrhyzae fungi on heavy metal uptake by soybeans. Soil Biol. Biochem.22:865-869.
    [27" class="description-paragraph] 27. Hiscox, J.D., and Israelstam G.F. 1979. A method for the extraction of chlorophyll from leaf tissue without maceration. Can. J. Bot. 51, 1332-1334. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein-dye binding. Anal. Biochem.72:248-258.
    [28" class="description-paragraph] 28. Hyodo, H. 1991. Stress/wound ethylene. In The Plant Hormone Ethylene, eds. A.K. Mattoo and J.C. Suttle, pp. 65-80, CRC Press, Boca Raton.
    [29" class="description-paragraph] 29. Jackson, M.R. 1991. Ethylene in root growth and development. In The plant hormone ethylene. Edited by A.K.Matto, and C.S.Suttle. CRC Press, Boca Raton, Florida. pp.151-189.
    [30" class="description-paragraph] 30. Jacobson, C.B., Pasternak, J.J. and Glick, B.R. 1994. Partial purification and characterization of the enzyme ACC deaminase from the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2. Can. J. Microbiol.40: 1019-1025.
    [31" class="description-paragraph] 31. Keberle, H. 1964. The biochemistry of desferrioxamine and its relation to iron metabolism. Ann. N. Y. Acad. Sci. 119: 758-768.
    [32" class="description-paragraph] 32. Killham, K. and Firestone, M. K. 1984. Vesicular arbuscular mycorrhyzal mediationof grass responce to acidic and heavy metal deposition. Plant Soil 72:39-48.
    [33" class="description-paragraph] 33. Kumar, P.B.A.N., Dushenkov,V., Motto, H., and Raskin, 1. 1995. Phytoextraction: the use of plants to remove heavy metals from soil. Environ. Sci. Technol.29:1232-1238.
    [34" class="description-paragraph] 34. Lynch, J.M. (1990). The Rhizosphere. Wiley-Interscience, Chichester) England.
    [35" class="description-paragraph] 35. Mergeau, M., Nies, D. Schlegel, H.G., Gerits,J., Charles, P., and van Gijsegem, F. 198 Alcaligenes eutrophus CH34 is a facultative chemolitotroph with plasmid-bound resistance to heavy metals. J. Bacteriol. 162:328-334.
    [36" class="description-paragraph] 36. Mehra, A., and Farago, M.E. 1994. Metal ions and plant nutrition. In Plants and chemical elements. Biogeochemistry, uptake, tolerance and toxocity. Edited by M.E. Fara o. VCH, Weinheim. pp.32-36.
    [37" class="description-paragraph] 37. Miller, J.H. 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
    [38" class="description-paragraph] 38. Mishra, D. and Kar, M. 1974. Nickel in plant growth and metabolism. Bot. Rev. 40:395-452.
    [39" class="description-paragraph] 39. Raaska, L., Viikari, L. and Mattila-Sandholm, T. 1993. Detection of siderophores in growing cultures of Pseudomonas spp., J. Ind. Microbiol. II:181-189.
    [40" class="description-paragraph] 40. Reeves, R.D. 1988. Nickel and zinc accumulation by species of Thlaspi L., Cochlearia L., and other genera of the Brassicaceae. Taxon 37:309-318.
    [41" class="description-paragraph] 41. Reid, C.P.P., Szaniszlo, P.J., and Crowley, D.E. 1986. Siderophore involvement in plant iron nutrition. In Iron siderophores and plant diseases. Edited by T.R. Swinburne. Plenum Press, New York. pp. 29-42.
    [42" class="description-paragraph] 42. Salt, D.E., Blaylock, M., Kumar, N.P.B.A., Dushenkov,V., Ensley, B.D., Chet, I., and Raskin, I. 1995. Phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants. BiolTechnology 13:468-474.
    [43" class="description-paragraph] 43. Schmidt, T., and Schlegel, H.G. 1989. Nickel and cobalt resistance of various bacteria isolated from soil and highly polluted domestic and industrial wastes. FEMS Microbiol. Ecol. 62:315-328.
    [44" class="description-paragraph] 44. Schwyn, B., and Neilands, J.B. 1987. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 160:47-56.
    [45" class="description-paragraph] 45. Tam, P.C.F. 1995. Heavy metal tolerance by ectomycorrhyzal fungi and metal amelioration by Pisolithus tinctorium. Mycorrhyza 5:181-187.
    [46" class="description-paragraph] 46. Turner) A.P. 1994. The responces of plants to heavy metals. In Toxic metals in soil-plant systems. Edited by S.M.Ross. John Wiley & Sons, Chichester.pp.153-187.
    [47" class="description-paragraph] 47. Van Loon, L.C. 1984. Regulation of pathogenesis and symptom expression in diseased plants by ethylene. In Ethylene: Biochemical, Physiological and Applied Aspects, eds. Y. Fuchs and E. Chalutz, pp. 171-180, Martinus Nijhoff/Dr W. Junk, The Hague.
    [48" class="description-paragraph] 48. Wang, Y., Brown, H.N., Crowley, D.E., and Szaniszlo, P.J. 1993. Evidence for direct utilization of a siderophore, ferroxamine B, in axenically grown cucumber. Plant Cell Environ. 16:579-585.
    [49" class="description-paragraph] 49. Weckx, J., Vangronsveld, J., and Clijster, H. 1993. Heavy metal induction of ethylene production and stress enzymes. I. Kinetics of responce. In Cellular and molecular aspects of the plant hormone ethylene. Edited by J.C. Pech, A. Latche, and C. Balaque. Kluyver Academic Press, Holland. pp. 239-238.
    [50" class="description-paragraph] 50. Wilkins, D.A. 1978. The measurement of tolerance to edaphic factors by means of root growth. New Phytol. 80:623-633.
    [51" class="description-paragraph] 51. Yang, X., Baligar, V.C., Martens, D.C., and Clark, P.B. 1996. Plant tolerance to nickel toxicity: II Nickel effect on influx and transport of mineral nutrients in four plant species. J. Plant Nutrit. 19:265-279. Table 1. Characteristics and heavy metal content of the soil sample used for the isolation of K. ascorbata SUD165. Metal or Characteristic Value Barium, mg/kg dry weight soil 35.2 Cadmium, mg/kg dry weight soil 0.25 Chromium, mg/kg dry weight soil 36.0 Cobalt, mg/kg dry weight soil 57.9 Copper, mg/kg dry weight soil 219. Lead, mg/kg dry weight soil15.1 Nickel, mg/kg dry weight soil 752. Silver, mg/kg dry weight soil 0.76 Vanadium, mg/kg dry weight soil33.4 Zinc, mg/kg dry weight soil 94.5 Loss on ignition, %2.88 Moisture content, % 67.3 Total carbon, % 19.3 Total inorganic carbon, % <0.01 pH 5.58 Table 2. The effect of K. ascorbata SUD165 on canola root elongation in gnotobiotic growth pouches. In experiements 4~6, the values of two treatments differ significantly from each other (P<0.001 ) in all three instances. Data were analyzed by ANOVA with 55-60 seeds tested for each value reported. The absorbance at 600 nm of the bacterial suspension was 0.025. Experiment Strain Light level mmo1/M2/sec Root length t 1 S E1 none 12.949.2 t 3.3 SUD165 12.9 45.3 t 2.8 2 none 12.9 44.6 t 3.0 SUD165 12.9 45.9 t 3.0 3 none 12.9 42.6 t 2.1 SUD165 12.9 43.1 t 1.6 4 none 0.944.7 t 3.8 SUD165 0.959.2 t 3.9 5 none 0.936.1 t 2.5 SUD165 0.9 47.5 t 3.0 6 none 0.945.5 t 3.1 SUD165 0.955.7 t 2.3 Table 3. Effect of K. ascorbata SUD165 on toxicity of nickel to canola seedlings grown in gnotobiotic growth pouches. Cells A = K. ascorbata SUD165 cells diluted to an absorbance at 600 nm of 0.025; Cells B = an absorbance of of 0.50. The values of the different treatments differ significantly from the untreated control and from each other (P<0.001 ). Each value (including the seeds grown in the absence of nickel, i.e. Tolerance index = 1.0) reflects the average of the response of 5060 seeeds. Tolerance index Experiment Oraan~Ni++1. mM Ni++ only Ni++ + cellsNi++ + A cells B 1 Shoots 1 0.48 0.75 n.d. Roots1 0.57 0.85n.d. 2 Shoots 2 0.22 0.490.71 Roots2 0.11 0.340.54 3 Shoots 2 0.22 0.47 n.d. Roots2 0.08 0.32n.d. 4 Shoots 1 0.44 0.710.81 Roots1 0.49 0.730.87 Table 4. Effect of K. ascorbata SUD165 on toxicity of nickel to canola seedlings growl' in soil in pots. The [Ni++] was 6 mM and the K. ascorbata SUD165 cells were diluted to an absorbance at 600 nm of 0.50. The values of different treatment differ significantly (P<0.001 ) frorn untreated control and from each other. Data were analyzed by ANOVA with seeds in each group. Each value reflects the average of the response of 50-60 seeeds. n.d.not determined. Tolerance index Experiment Organ NI++ only Ni++ + cells 1 Shoots 0.12 0.18 1 Roots 0.23 0.33 2 Shoots 0.18 0.14 22 Roots 0.23 0.36 Table 5. The effect of inoculation with K.ascorbata SUD165 cells on Ni++ toxicity to tomato seeds development in pots. The K. ascorbata SUD165 cells were diluted to an absorbance at 600 nm of 0.50. The values of different treatment differ significantly (P<0.001 ) from untreated control and from each other. Each value reflects the average of the response of 50-60 seeeds. Tolerance index Experiment Organ (Ni++], m'M Ni++ only Ni+++ cells 1Roots 3 0.57 0.80 1 Shoots 3 0.54 0.70 2 Roots 4 0.35 0.56 2 Shoots 4 0.42 0.59 Table 6. 63Ni++ accumulation by canola seedlings in the presence and absence of K.ascorbata SUD165 cells. The K. ascorbata SUD165 cells were diluted to an absorbance at 600 nm of 0.50. The data is expressed as pmoles of ssNici2 per mg dry weight of plant tissue.Each number represents an average of five experiments in which the total radioactivity 1 SEM) incorporated into 10-15 roots or shoots was measured. Or~c an Ni++ only Ni++ + cells Roots 1430. 183.7 1370. t 380. Shoots 178. 42.3 174.164.5 o ~d a~ o b cC a~ -- _ _. C O O O O O O U ~~ ~ O O O O O O O O. O ~ O ~o ~ .o o A~ ' . ~~ ~ ,~ ~ ~ d N ~ ~ M v0 0Oy N 0 ~ ~O v1v1 M N '-"w tn . ~ _ ~O ~ V7 ~~n C __ O ~ O ~ DM a ~O 'H c M h ~ ~ ~ O O O v.r '+' M CC h U cC ~ ~ ~n ~ ~ ~ o o N O ~ ~ v'~~ .--U c~~ ~'!7 N"d ~ _U U ~ O N 01 ~ l~ 00 O N o0 v1 ~ O ~ O O Q. ~ ~ ~ ~ Aw U _ ~(~ it~ M O ~ lp V1 zo ~.3 E-' ~-' V1 ~ UN~ v1 ~O .~C~n vO .~G1 . U _ r~N U _O C O y =p ~O O 'C 'C ~ v1 N O '~'C C v1 N a .- ~c ~~w U N V ~O~ O o0p~ _ a C ~ ~o 0o p M ~ _ o ; 0 0 . ~ c~C p ~n ~' ~ 'v 'c o 0 o c~ O r~ C C v1 N " C ,~ U ~ '" U '~ C~ ~ C O N~ M et V1 O O O O O O~ n ~M M l1 00 O O M M e~'~!1N W U ~1 a0 00 '~; ~ c c D 3 o c ~ = 3 . en a~ ~ c 'v ayn ~ ~ n. . ou ~ % 0 ~ ~ '~ ~ ~ E ~ E- a, a, -c a _ bU . ~i O T N G, CG bD D E O . ~ 6~ U O N C O~ _a~ U .a.. ~ S. ~ s ~ C ~N ~ N.-U ~ 3 . . ~ E= '~ 0 z z ~ v '~ o ~ o ~n o ~" ~ N N M
    权利要求:
    Claims (27)
    [1] 1. An agent which ameliorates apparent toxicity of one or more heavy metals to a plant.
    [2] 2. The agent of claim 1 which is a bacterium.
    [3] 3. The agent of claim 2 which is known as K. ascorbata SUD 165.
    [4] 4. The agent of claim 2 which is known as K. ascorbata SUD 165/26.
    [5] 5. An agent which ameliorates apparent toxicity of Ni++ to a plant.
    [6] 6. The agent of claim 5 which is a bacterium.
    [7] 7. The agent of claim 6 known as K. ascorbata SUD 165.
    [8] 8. The agent of claim 6 known as K. ascorbata SUD 165/26.
    [9] 9. Siderophore producing, plant growth promoting bacteria which ameliorate apparent toxicity of one or more heavy metals to a plant.
    [10] 10. The bacteria of claim 9 known as K. ascorbata SUD 165.
    [11] 11. The bacteria of claim 9 known as K. ascorbata SUD 165/26.
    [12] 12. Siderophore producing, plant growth promoting bacteria which ameliorate apparent toxicity of Ni++ to a plant.
    [13] 13. The bacteria of claim 12 known as K. ascorbata SUD 165.
    [14] 14. The bacteria of claim 12 known as K. ascorbata SUD 165/26.
    [15] 15. Siderophore producing, plant growth promoting bacteria which ameliorate apparent toxicity of one or more heavy metals to a plant and reduce the amount of ethylene that evolves from said plant.
    [16] 16. The bacteria of claim 15 known as K. ascorbata SUD 165.
    [17] 17. The bacteria of claim 15 known as K. ascorbata SUD 165/26.
    [18] 18. Siderophore producing, plant growth promoting bacteria which ameliorate apparent toxicity of Ni++ to a plant and reduce the amount of ethylene that evolves from said plant.
    [19] 19. The bacteria of claim 18 known as K. ascorbata SUD 165.
    [20] 20. The bacteria of claim 18 known as K. ascorbata SUD 165/26.
    [21] 21. Siderophore producing, plant growth promoting bacteria known as K.ascorbata SUD 165/26 which ameliorate apparent toxicity of one or more heavy metals to a plant and reduce the amount of ethylene that evolves from said plant.
    [22] 22. The bacteria of claim 24 wherein said plant is a seed.
    [23] 23. The bacteria of claim 21 or 22 where the heavy metal is Ni++ and the plant is selected from the group comprised of tomato and canola.
    [24] 24. A method to ameliorate apparent toxicity of one or more heavy metals to a plant and reduce the amount of ethylene that evolves from said plant which consists of adding a sufficient amount of siderophore producing, plant growth promoting bacteria to said plant.
    [25] 25. The method of claim 24 wherein the siderophore producing, plant growth promoting bacteria is K. ascorbata SUD 165/26.
    [26] 26. The method of claim 25 wherein said bacteria is added to seeds.
    [27] 27. The method of claim 24 wherein said plant is chosen from the group comprising canola and tomato.
    类似技术:
    公开号 | 公开日 | 专利标题
    Burd et al.1998|A plant growth-promoting bacterium that decreases nickel toxicity in seedlings
    Ma et al.2015|The hyperaccumulator Sedum plumbizincicola harbors metal-resistant endophytic bacteria that improve its phytoextraction capacity in multi-metal contaminated soil
    Zhang et al.2011|Characterization of lead-resistant and ACC deaminase-producing endophytic bacteria and their potential in promoting lead accumulation of rape
    Sharma et al.2003|Plant growth-promoting bacterium Pseudomonas sp. strain GRP3 influences iron acquisition in mung bean |
    Van Overbeek et al.1995|Root exudate-induced promoter activity in Pseudomonas fluorescens mutants in the wheat rhizosphere
    Loper et al.1994|A biological sensor for iron available to bacteria in their habitats on plant surfaces
    Rajput et al.2013|Salt-tolerant PGPR strain Planococcus rifietoensis promotes the growth and yield of wheat | cultivated in saline soil
    Loper1988|Role of fluorescent siderophore production in biological control of Pythium ultimum by a Pseudomonas fluorescens strain.
    Ma et al.2011|Inoculation of endophytic bacteria on host and non-host plants—effects on plant growth and Ni uptake
    Siripornadulsil et al.2013|Cadmium-tolerant bacteria reduce the uptake of cadmium in rice: potential for microbial bioremediation
    Davison1988|Plant beneficial bacteria
    Loper et al.1987|Lack of evidence for the in situ fluorescent pigment production by Pseudomonas syringae pv. syringae on bean leaf surfaces.
    Ma et al.2009|Improvement of plant growth and nickel uptake by nickel resistant-plant-growth promoting bacteria
    Karagöz et al.2012|Characterization of plant growth-promoting traits of bacteria isolated from the rhizosphere of grapevine grown in alkaline and acidic soils
    Yahaghi et al.2018|Isolation and characterization of Pb-solubilizing bacteria and their effects on Pb uptake by Brassica juncea: implications for microbe-assisted phytoremediation
    Raiesi et al.2006|Interactions between phosphorus availability and an AM fungus | and their effects on soil microbial respiration, biomass and enzyme activities in a calcareous soil
    Rebah et al.2002|Wastewater sludge as a substrate for growth and carrier for rhizobia: the effect of storage conditions on survival of Sinorhizobium meliloti
    Anandham et al.2008|Potential plant growth promoting traits and bioacidulation of rock phosphate by thiosulfate oxidizing bacteria isolated from crop plants
    Sarathambal et al.2017|The effect of plant growth-promoting rhizobacteria on the growth, physiology, and Cd uptake of Arundo donax L.
    Pishchik et al.2009|Interactions between plants and associated bacteria in soils contaminated with heavy metals
    Gamalero et al.2012|19 Plant Growth-Promoting Bacteria and Metals Phytoremediation
    Wani et al.2007|Cadmium, chromium and copper in greengram plants
    EP0256889A1|1988-02-24|Plant growth-promoting rhizobacteria for agronomic, nonroot crops
    Subrahmanyam et al.2018|Vigna radiata var. GM4 plant growth enhancement and root colonization by a multi-metal-resistant plant growth-promoting bacterium Enterobacter sp. C1D in Cr |-amended soils
    Jing et al.2012|Isolation and characterization of heavy-metal-mobilizing bacteria from contaminated soils and their potential in promoting Pb, Cu, and Cd accumulation by Coprinus comatus
    同族专利:
    公开号 | 公开日
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2004-02-18| EEER| Examination request|
    2008-02-18| FZDE| Dead|
    优先权:
    申请号 | 申请日 | 专利标题
    US7509998P| true| 1998-02-18|1998-02-18||
    US60/075,099||1998-02-18||
    [返回顶部]