WO2002045513A2 - Bacterial inoculants for enhancing plant growth - Google Patents
Bacterial inoculants for enhancing plant growth Download PDFInfo
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- WO2002045513A2 WO2002045513A2 PCT/US2001/046524 US0146524W WO0245513A2 WO 2002045513 A2 WO2002045513 A2 WO 2002045513A2 US 0146524 W US0146524 W US 0146524W WO 0245513 A2 WO0245513 A2 WO 0245513A2
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
- A01N43/40—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom six-membered rings
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/08—Magnoliopsida [dicotyledons]
- A01N65/20—Fabaceae or Leguminosae [Pea or Legume family], e.g. pea, lentil, soybean, clover, acacia, honey locust, derris or millettia
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/40—Liliopsida [monocotyledons]
- A01N65/44—Poaceae or Gramineae [Grass family], e.g. bamboo, lemon grass or citronella grass
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/22—Klebsiella
Definitions
- Bacillus cereus AS4-12 (ATCC No. 55609) has been found to increase the efficacy in fostering the growth and establishment of alfalfa plants in the upper mid-western United States (See U.S. Patent No. 5,552,138).
- strains of Pantoea agglomerans may also be able to increase the yield of legumes and inhibit the growth of phytopathogenic fungi. Hoflich and Ruppel, "Growth stimulation of pea after inoculation with associative bacteria," Microbiol. Res., 149:99-104 (1994). P.
- agglomerans (formerly Enterobacter agglomerans) is a Gram-negative Enterobacterium often found associated with plants, water, soil, or foodstuffs. P. agglomerans is also a diazotroph, and able to fix nitrogen in both pure culture and in association with wheat.
- Merbach et al. "Dinitrogen fixation of microbe-plant associations as affected by nitrate and ammonium supply,” Isotopes Environ. Health Stud., 34:67-73 (1998). It has also been reported to produce two auxins and two cytokines in pure culture. Scholz et al., "Development of DAS-ELISA for some selected bacteria from the rhizosphere," Mariabl. Mikrobiol.
- Klebsiella pneumoniae is also a member of the family Enterobacteriaceae and a known nitrogen fixing bacterium, i.e. able to convert atmospheric nitrogen into ammonium.
- K. pneumoniae is a free-living soil bacterium and unlike other nitrogen- fixing bacteria, such as Rhizobium, K. pneumoniae does not participate in symbiotic interactions with leguminous plants.
- K. pneumoniae has also not yet been shown to be effective in enhancing the growth of cereal grasses.
- biological control agents are able to increase agricultural productivity and efficiency are diverse, and will vary depending upon the unique characteristics of each particular agent. It is believed, for example, that certain bacteria are able to control root rot in plants by competing with fungi for space on the surface of the plant root. It is also believed that competition between various bacterial strains in a plant's native microflora may stimulate root growth and increase the uptake of mineral nutrients and water to enhance plant yield. Alternatively, toxins produced by certain bacterial species are believed to facilitate plant growth by controlling bacterial species pathogenic to the plant. Bacterially produced antibiotics are an example of such toxins.
- the present invention is summarized as a method for enhancing the growth of a plant using an inoculum comprising an effective quantity of bacteria selected from the group consisting of Klebsiella pneumoniae 342, Klebsiella pneumoniae zmvsy, Pantoea agglomerans PlOl, Pantoea agglomerans PI 02, Herbaspirillum seropedicae 2 A, Herbaspirillum serope ⁇ icae Z152, Gluconacetobacter ⁇ iazotrophicus PA15, and any mutations thereof which retain the ability to enhance plant growth.
- the present invention also includes the bacterial inoculant of the above method, and a plant seed coated with the bacterial inoculant.
- the present invention includes a method for identifying strains of Pantoea agglomerans and Klebsiella pneumoniae which have the ability to enhance the growth of cereal grasses. Also included is an inoculum comprising the bacterial strains identified by said method and a method for enhancing the growth of cereal grasses using said bacterial strains. The present invention also includes a plant seed coated with the inoculum.
- the present invention is further characterized in that novel bacterial strains capable of enhancing the growth of a plant have been isolated from the environment.
- novel bacterial strains capable of enhancing the growth of a plant have been isolated from the environment. These strains include Herbaspirillum serope ⁇ icae 2A (ATCC No. PTA- 2742), Pantoea agglomerans PlOl (ATCC No. PTA 2744), Pantoea agglomerans PI 02 (ATCC No. PTA 2740), Klebsiella pneumoniae 342 (ATCC No. PTA-2743), Klebsiella pneumoniae zmvsy (ATCC No. PTA-2741), and mutations thereof which retain the ability to enhance the growth of plants.
- the present invention is directed toward certain bacterial strains and inoculants capable of enhancing the growth of plants.
- the bacterial strains utilized in the present invention include Herbaspirillum serope ⁇ icae 2A, Herbaspirillum serope ⁇ icae Z152, Pantoea agglomerans ⁇ ⁇ PlOl, Pantoea agglomerans PI 02, Klebsiella pneumoniae 342, Klebsiella pneumoniae zmvsy, Gluconacetobacter ⁇ iazotrophicus PA 15, and any mutations thereof which retain the ability to enhance the growth of plants.
- strains of the bacterium P. agglomerans and K. pneumoniae which are able to enhance the growth of cereal grasses as identified by the method described below.
- the above bacterial strains shall sometimes be referred to collectively as "enhancing" bacteria.
- Bacterial strains H. serope ⁇ icae 2A, P. agglomerans PlOl, P. agglomerans PI 02, K. pneumoniae 342, and K. pneumoniae zmvsy are believed to be new to the world, and were deposited with the American Type Culture Collection ("ATCC"), Manassas, VA 20110-2209 U.S.A., on November 30, 2000.
- ATCC American Type Culture Collection
- the bacterial strain H serope ⁇ icae 2A was isolated from a maize plant cultivated in Hancock, Wisconsin during the summer of 1999, and has been given the ATCC Patent Deposit Designation PTA-2742.
- agglomerans PI 02 were isolated from switch grass plants collected from a remnant prairie near Plover, Wisconsin, and have been given the ATCC Patent Deposit Designation PTA 2744 and PTA 2740, respectively.
- K. pneumoniae 342 was isolated from a very nitrogen efficient line of maize from Mexico cultured in a greenhouse in Madison, Wisconsin, and has been given the ATCC Patent Deposit Designation PTA-2743.
- K pneumoniae zmvsy was isolated in 1994 from a maize plant grown on a farm in Madison, Wisconsin, and has been given the ATCC Patent Deposit Designation PTA-2741.
- Bacterial strains H. serope ⁇ icae Z152 (ATCC No. 35894) and G. ⁇ iazotrophicus PA15 (ATCC No. 49037) were both previously known and available from the ATCC, but never shown to enhance the growth of cereal grasses. Both bacterial strains were isolated in the late 1980s from sugarcane in Brazil. [0019] It is anticipated that certain mutants of the enhancing bacteria may also enhance plant growth comparable to the non-mutated forms set forth above. Mutants of the enhancing bacteria may include both naturally occurring and artificially induced mutants. Certain of these mutants will be found to enhance cereal grasses or legumes using the plant enhancement assay described below.
- Others mutants may be induced by subjecting the enhancing bacteria to known mutagens, such as N-methyl- nitrosoguanidine, using conventional methods. Similar mutants have been made from useful Bacillus cereus strains such as UW85 (ATCC No. 53522) and AS4-12 (ATCC No. 55609) as described in U.S. Patent No. 4,877, 738 and U.S. Patent No. 5,552,138, respectively, the disclosure of which is hereby incorporated by reference. [0020] The data set forth below in the Examples demonstrate that other strains of the bacterium P. agglomerans and K. pneumoniae will also be effective in enhancing the growth of cereal grasses when used in accordance with the present invention. These strains may be isolated using methods commonly known in the art for isolating free- living organisms from the environment, and their ability to enhance plant growth may be verified using any one of many plant enhancement assays.
- the following is a disclosure of one such plant enhancement assay whereby a bacterial isolate, or the like, may be tested for its ability to enhance the growth of a cereal grass next to which it is placed.
- the seed or seedling of the cereal grass to be enhanced is planted in a planting medium and watered with a nutrient solution.
- the planting medium may be a damp soil, vermiculite in water, an agar-based formulation, or any other planting medium in which the seed or seedling will grow and develop.
- the bacterial isolate is placed at least in the immediate vicinity of the seed or seedling.
- Such placement shall be understood to be in the "immediate vicinity" of the seed or seedling if the bacterial isolate or any soluble exudate of a bacterium being tested will be in actual contact with the germinating seedling. After a time sufficient for seedling growth, seedlings developing from the planted seed may be evaluated for visual evidence of enhanced growth when compared to controls.
- the bacterial inoculants of the present invention act through an unknown mechanism to enhance plant growth. While the mechanism by which these inoculants enhance plant growth is not understood, it is possible that the mechanism involves an antagonistic action by the enhancing bacterium on other organisms which may inhibit and/or retard the germination and growth of the plant seedling. The method of action may alternatively involve a symbiotic relationship of some unknown type. [0023] It is broadly intended within the scope of the present invention that the bacterial inoculant of the present invention be inoculated into the soil with plant seeds so that a culture of the enhancing bacteria will develop in the root system of the plant as it grows.
- the inoculant preferably diluted with a suitable extender or carrier, either be applied to the seeds prior to planting or introduced into the seed furrows when the seeds are planted.
- the bacterial inoculant so delivered may be any viable bacteria culture capable of successful propagation in the soil.
- the bacterial inoculant be applied to the seeds through the use of a suitable coating mechanism or binder prior to the seeds being sold into commerce for planting.
- the process of coating seed with such an inoculum is generally well known to those skilled in the art.
- the enhancing bacteria may be mixed with a porous, chemically inert granular carrier as described by U.S. Patent No. 4,875,921 (incorporated herein by reference).
- the bacterial inoculant may be prepared with or without a carrier and sold as a separate inoculant to be inserted directly into the furrows into which the seed is planted. The process for inserting such inoculants directly into the furrows during seed planting is also generally well known in the art.
- the enhancing bacteria may also be obtained in a substantially pure culture.
- a "substantially pure" culture shall be deemed to include a culture of bacteria containing no other bacterial species in quantities sufficient to interfere with the replication of the culture or be detected by normal bacteriological techniques.
- the enhancing bacteria is preferably diluted with a suitable carrier or extender so as to make the culture easier to handle and to provide a sufficient quantity of material so as allow easy human handling.
- a peat based carrier may be used as described by Bosworth et al, "Alfalfa yield response to inoculation with recombinant strains of Rhizobium meliloti carrying an extra copy of act and/or modified nifK expression," Appl. Environ. Microbiol., 60:3815-3832 (1994), incorporated herein by reference.
- perlite, vermiculite and charcoal materials are suitable carrier substances. It is believed that many other non-toxic and biologically inert substances of dried or granular nature are also capable of serving as carriers for the enhancing bacteria.
- the density of inoculation of these bacterial cultures onto seed or into the furrows should be sufficient to populate the sub-soil region adjacent to the roots of the plant with viable bacterial growth.
- An effective amount of bacterial inoculant should be used.
- An effective amount is that amount sufficient to establish sufficient bacterial growth so that the yield from the plant is increased.
- the enhancing bacterial strains are isolated from the roots of exceptionally vigorous plants grown under conventional cultivation practices. Once isolation of the strains was made, the bacterial culture had to be cultivated to generate sufficient quantities of material for proper seed treatment. It has been discovered here that the inoculation of various cereal grasses with the enhancing bacterial strains results in significantly improved growth of the cereal grass plants. As will be appreciated by any person skilled in plant husbandry, the rate of growth or improvement in growth of any given crop is subject to many variables. It has been found here, however, that the co-cultivation of the bacterial inoculant of the present invention with cereal grasses is of significant advantage in at least some typical field conditions.
- the bacterium grows in cultivation along with the plants and should continue to exhibit its beneficial effect on the plant throughout the agricultural season. This is in strong contrast to chemical growth agents or fungicides which must be retreated periodically to have a continuing effect on inhibition of the fungus in question or to help improve the plant growth throughout its life cycle. Since the bacterial inoculant of the present invention can be inoculated onto the seeds using a dry or wet formulation, the application of this technique is relatively simple to the farmer since the seeds can be inoculated prior to distribution. In this way, a significant economic advantage is achievable. [0032] The following non-limited examples are intended to illustrate the present invention.
- the bacterial strains which make up the bacterial inoculants of the present invention were isolated (or obtained from the ATCC in the case of Gluconacetobacter ⁇ iazotrophicus PA15 and Herbaspirillum serope ⁇ icae Z152) and grown in culture on petri dishes at 28°C to create crop inoculating propagules.
- the culturing media used for Klebsiella pneumoniae 342, Klebsiella pneumoniae zmvsy, Pantoea agglomerans PlOl and Pantoea agglomerans PI 02 was Luria-Bertani medium.
- the medium used for Gluconacetobacter ⁇ iazotrophicus PA15 was AcD medium, which contains per liter: 0.64 g K 2 HPO 4 , 0.16 g KH 2 PO 4 , 0.2 g MgSO 4 JH 2 O, 0.2 g NaCl, 0.05 g CaSO 4 -2H 2 O, 20 g sucrose, 2 mg NaMoO 4 , 3 mg FeSO 4 , 2 g MES buffer, 1 g malic acid, and 0.1 g yeast extract. This medium was adjusted to pH 6.5 prior to autoclaving.
- Herbaspirillum serope ⁇ icae 2A and Herbaspirillum serope ⁇ icae Z152 were cultured on BSM medium as described by Bergersen et al., "The Growth of Rhizobium in Synthetic Medium," Aust. J. Biol. Sci., 14:349-360 (1961). All media contained 15 g/L of agar.
- Wheat seeds from three cultivars were inoculated with the bacterial propagules to determine each bacterial strains' ability to affect plant growth. Seeds were first surface sterilized using 70% ethanol for 30 seconds and 10% bleach for 2 minutes, followed by 6 washes with sterile water. A cell suspension of approximately 10 cells/ml was then added to the seeds for a few hours prior to planting to inoculate the wheat seeds.
- Inoculated and uninoculated wheat seeds were planted approximately 1 cm deep in pots containing 2 liters of a T.l sand/vermicullite mixture. Plants were watered with a nutrient solution described by Chelius et al., "Immunolocalization of dinitrogenase reductase produced by Klebsiella pneumomae in association with Zea mays L.," Appl. Environ. Microbiol., 66:783-787 (2000), containing all essential nutrients except nitrogen. Four seeds were planted per pot which were thinned to two plants per pot after two weeks.
- Maize seeds were inoculated prior to planting by coating 1 kg of seeds with a 125 mL bacterial suspension in 8 grams of peat (0.566 gCaCO 3 , 0.288 g charcoal, 10 mL 40% Vitalive (Research Seeds Inc., St. Joseph, MO, USA) in a plastic bag.
- the cell suspension contained approximately 8 x 10 8 cells per mL to ensure an inoculum density of 10 8 cells per seed. Cell number per seed was verified after inoculation by suspending seeds in water and plating various dilutions on the appropriate medium.
- the seeds included seeds from maize inbred lines B73 and Mo 17; the hybrid B73 x Mo 17; two lines with a significant teosinte background, 1/4 teosinte background, and 1/2 teosinte background; and various hybrid lines from Pioneer.
- Seeds were planted within 48 hours of inoculation in rows thirty feet in length, with 30 seeds planted per row. Rows were approximately 36 inches apart. Weeds were controlled chemically by standard agricultural practices for the region. Fertilizer was applied prior to planting at rates of 17.2 g m " P O 5 and 51.5 g m " ⁇ K 2 O. Where nitrogen was applied, it was applied at a rate of 224 kg NH NO 3 ha "1 prior to planting. An uninoculated control was included with each maize line. [0041] After the growing season, the treated com was harvested and grain weight and moisture measured for each plot. Yield was determined as grain yield in t ha '1 , standardized to 15.5% moisture.
- Grain was harvested with a combine equipped with a HarvestMaster data logger (HarvestMaster, Inc., Logan, UT, USA) for the determination of weight and moisture levels of the grain from each plot.
- the values shown in Table 2 below illustrate the increased yield per acre (or hectare), after correcting for moisture levels. Grain yield is expressed in metric t ha "1 . Only those combinations where statistically significant increases were obtained are listed. TABLE 2
- Example 2 Arlington Agricultural Research Station (Arlington, Wisconsin) in a manner similar to Example 2. After the growing season, the treated corn was harvested and grain weight and moisture measured for each plot. The values shown in Table 3 below illustrate the increased yield per acre (or hectare), after correcting for moisture levels. Grain yield is expressed in metric t ha "1 . Only those combinations where statistically significant increases were obtained are listed.
- Example 1 above to determine each bacterial strains' ability to affect the growth of rice plants. Seeds were first surface sterilized using 70% ethanol for 30 seconds and 10% bleach for 2 minutes, followed by 6 washes with sterile water. A cell suspension of approximately 10 cells/ml was then added to the seeds to inoculate the seeds for a few hours prior to planting.
- Inoculated and uninoculated rice seeds were planted approximately 1 cm deep in pots containing 2 liters of a 1:1 sand/vermicullite mixture. Plants were watered with a nutrient solution described by Chelius et al., "Immunolocalization of dinitrogenase reductase produced by Klebsiella pneumoniae in association with Zea mays L.," Appl. Environ. Microbiol., 66:783-787 (2000), containing all essential nutrients except nitrogen. Four seeds were planted per pot which were thinned to two plants per pot after two weeks. • ⁇ •
- the rice shoots of the plants were harvested, placed in a paper bag, and dried for one week in driers at about 80°C. After drying, the plants were weighed. The values shown in Table 5 below were obtained by comparing the average dry weight of the treated plants to the average dry weight of untreated plants.
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EP01996114A EP1339286A2 (en) | 2000-12-04 | 2001-12-03 | Bacterial inoculants for enhancing plant growth |
CA002429751A CA2429751A1 (en) | 2000-12-04 | 2001-12-03 | Bacterial inoculants for enhancing plant growth |
AU2722802A AU2722802A (en) | 2000-12-04 | 2001-12-03 | Bacterial inoculants for enhancing plant growth |
AU2002227228A AU2002227228B2 (en) | 2000-12-04 | 2001-12-03 | Bacterial inoculants for enhancing plant growth |
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US25113700P | 2000-12-04 | 2000-12-04 | |
US60/251,137 | 2000-12-04 |
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Also Published As
Publication number | Publication date |
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AU2002227228B2 (en) | 2007-05-31 |
US20020142917A1 (en) | 2002-10-03 |
WO2002045513A3 (en) | 2002-11-21 |
EP1339286A2 (en) | 2003-09-03 |
CA2429751A1 (en) | 2002-06-13 |
US7393678B2 (en) | 2008-07-01 |
AU2722802A (en) | 2002-06-18 |
US20040116291A1 (en) | 2004-06-17 |
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