# Patent

## Title
Nitrogen-fixing bacterial inoculant for improvement of crop productivity and reduction of nitrous oxide emission

## Abstract

The present invention relates to methods of reducing chemical fertilizer usage and greenhouse gas nitrous oxide emission and to methods of improving plant growth rate and seed productivity in agriculture through the application of a novel artificially manufactured formula containing a nitrogen-fixing bacterium that efficiently colonizes non-legume plants in aerial parts and the root system. The bacteria inocula and methods are particularly suitable for plants in the genera Jatropha, Sorghum, Gossypium, Elaeis, Ricinus, Oryza and Manihot.

## Background
CROSS-REFERENCE OF THE RELATED APPLICATION
The present application is a 35 U.S.C.§371 National Phase Entry Application of PCT/SG2013/000112, filed 20 Mar. 2013, and designating the United States, which in turn claims priority from U.S. Provisional Application No. 61/613,579 filed 21 Mar. 2012. Each application is incorporated herein by reference in its entirety.


SEQUENCE SUBMISSION
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is entitled 2577211PCTSequenceListing.txt, created on 25 Jan. 2013 and is 49 kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
The present invention relates to methods of reducing chemical fertilizer usage and greenhouse gas nitrous oxide (N2O) emission and to methods of improving plant growth rate and seed productivity in agriculture through the application of a novel artificially manufactured formula containing a nitrogen-fixing bacterium that efficiently colonizes non-legume plants in aerial parts and the root system. The bacteria inocula and methods are particularly suitable for plants in the genera Jatropha, Sorghum, Gossypium, Elaeis, Oryza, Ricinus and Manihot. 
The rapid rise in fossil fuel price, the fast diminishment of global fuel and the concern about rapid global warming resulted from the accumulation of atmospheric greenhouse gas have served as the three catalysts in the recent biofuel boom (Chang, 2007). The environmental benefit of biofuel consumption is attributed from the widely believed outcome of reduced greenhouse gas (CO2) emission.
Recently, a new issue that has been brought up for debate regarding the benefit of biofuels. Nitrous oxide is produced naturally in the soil during the microbial processes of nitrification and denitrification. A significant percentage of the nitrogen fertilizer used in biofuel production is converted to reactive nitrogen N2O, a greenhouse gas which has 310 times the ability to trap heat in the atmosphere (Barton and Atwater, 2002). The benefit of CO2 mitigation through biofuel consumption will be cancelled out if the use of N-fertilizer is not controlled (Galloway et al., 2008; Melillo et al, 2009; Crutzen et al., 2008).
Application of nitrogen fertilizer has become an essential practice in modern agriculture as it is vital to maintain competitive crop productivity. It has been well known that legumes require much less input of nitrogen fertilizer owing to the presence of nitrogen-fixing structure called nodules, in which nitrogen-fixing microorganisms (diazotrophs), mostly belonging to species in the Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium genera (Jourand et al., 2004; Kaneko et al., 2000; Stacey et al., 1991; Gottfert et al., 2001), form a mutually beneficial symbiotic relationship with the bacteria supplying nitrogen source to the plants while drawing carbon source from the plants cells (Long, 1996; Young and Johnston, 1989). Some exception has been found recently. For example, Methylobacterium nodulans is the root nodule-inducing agent for some species in the genus Crotalaria, a leguminous plant (Renier et al., 2011; Jourand et al., 2005) and nitrogen-fixing nodules may develop in stem tissues of some legumes (Eaglesham and Szalay, 1983; Dreyfus and Dommergues, 1981). Frankia, Gram-positive soil bacteria also induce the formation of nitrogen-fixing root nodules in a few species in the Rosaceae family (Moir et al., 2011).
The formation of symbiotic nodules involves with complex genetic and chemical interactions between the diazotroph and host. For example, the symbiosis between Sinorhizobium meliloti and its plant hosts begins when the plant secretes an array of betaines and flavonoids into the rhizosphere: 4,4′-dihydroxy-2′-methoxychalcone, chrysoeriol, cynaroside, 4′,7-dihydroxyflavone, 6′′-O-malonylononin, liquiritigenin, luteolin, 3′,5-dimethoxyluteolin, 5-methoxyluteolin, medicarpin, stachydrine, trigonelline. These compounds attract S. meliloti to the surface of the root hairs of the plant where the bacteria begin secreting nodulation factor (Peters et al., 1986; Maxwell et al., 1989). As a consequence, symbiotic nodule-forming nitrogen-fixation is found almost exclusively in legume species. Bradyrhizobia and rhizobia share characteristics with plant growth promoting rhizobacteria (PGPR). Nodule inducing bacteria, like other PGPR, are capable of colonizing the roots of non-legume plants (Antoun et al., 1998).
Diazotrophs have been found in free-living bacteria, among which Azotobacter vinelandii is the best studied. An increasingly number of diazotrophic species, e.g., Azospirillum, Herbaspirillum, Burkholderia, Gluconacetobacter, have been reported to form atypical symbiotic relationship with plants (Zehr, 2011; Radiers et al., 2004; Pedraza, 2008). They often grow on the surface root system (rhizobacteria) although some are able to infect plant tissues (endophytic bacteria) and perform nitrogen fixation, which is also able promote plant growth. For example, U.S. Pat. No. 7,393,678 B2 describes strains of Klebsiella pneumonia, which colonize the root surface (Liu et al., 2011), are able to promote growth of cereals (wheat and corn) either in the presence or absence of chemical nitrogen application. However, only specially isolated mutant strains of Klebsiella pneumonia were able to perform satisfactory plant growth promoting function as the nitrogen-fixation activity is low in majority of strains. Perhaps, the best examples of endophytic nitrogen-fixation can be found in sugarcane and wild rice, with Herbaspirillum, Gluconacetobacter, Enterobacter, Azospirillum, Swaminathania and Acetobacter being the possible contributors of nitrogen-fixing species (Pedraza, 2008; Boddey et al., 1995; Baldani et al., 2002; Elbeltagy et al., 2001; Saravanan et al., 2008). Photosynthetic Bradyrhizobia are the natural endophytes of the African wild rice Oryza breviligulata while the intercellular colonization and growth-promoting effects of a Methylobacterium sp. was observed in common rice Oryza sativa L. Cv CO-43 although the later was believed to resulted from phytohormone secretion of the bacterium (Senthilkumar et al., 2009; Chaintreuil et al., 2000).
A study of Azospirilla indicates that, similar to nodulation and nitrogen-fixation in legumes, the endophytic colonization process is genetically controlled and exopolysaccharide production inhibits both endophytic colonization and nitrogen fixation in common wheat seedlings (Kennedy et al., 1997). In addition, a mutation that blocks exopolysaccharide synthesis prevents nodulation of peas by Rhizobium leguminosarum but not of beans by R. phaseoli (Borthakur et al., 1986). Therefore, the ability of a diazotroph to promote plant growth via nitrogen-fixation is not predictable from its performance in other plants.
Jatropha curcas is a small woody plant belonging to the Euphorbiaceae family. Several unique characteristics make it an ideal plant for biodiesel production (Fairless, 2007; Gaydou et al., 1982; Openshaw, 2000). These include the ability to grow on marginal land; low requirement for water; a non-food crop status and fast oil production in 0.5-2 years after planting compared to more than 3 years for oil palm. Accordingly, several Asian countries, particularly Indonesian and India, have made ambitious plans to promote jatropha plantation. Several other plants have also attracted strong interest as alternative crop for biofuel production. These include castor bean, sorghum, and sweet sorghum.
As Jatropha is targeted to marginal land where soil nutrient is low, the requirement for nitrogen fertilizer will be higher than other crops. Therefore, any technology that reduces nitrogen fertilizer usage will be highly desirable. Today there is little research on naturally occurring diazotrophs in Jatropha nor the application of these microbes for improvement of plant productivity. Similar situations can be found in other crops, such as Sorghum, castor bean and cassava.
SUMMARY OF THE INVENTION
The present invention relates to methods of reducing chemical fertilizer usage and greenhouse gas nitrous oxide emission and to methods of improving plant growth rate and seed productivity in agriculture through the application of a novel artificially manufactured formula containing a nitrogen-fixing bacterium that efficiently colonizes non-legume plants in aerial parts and root system. The bacteria inocula and methods are particularly suitable for plants in the genera Jatropha, Sorghum, Gossypium, Elaeis, Oryza, Ricinus and Manihot. 
In a first aspect, the present invention provides a biologically pure culture of a bacterial species selected from the Enterobacter, Methylobacterium, Sphingomonas, and Pleomorphomonas genera as described herein. In some embodiments, the bacterial species of the present invention are able to efficiently reduce atmospheric N2 to ammonia as evidenced by the acetylene reduction assay (AR assay) in planta. In some embodiments, the N2 reduction occurs on the leaf surface. In other embodiments, the N2 reduction occurs inside the leaf surface. In additional embodiments, the N2 reduction occurs on the surface of the roots or root system. In further embodiments, the N2 reduction occurs inside the root tissue. In some embodiments, the bacterial species is able to self-propagate efficiently on the leaf surface, root surface or inside plant tissues without inducing a noticeable plant defense reaction, such as cell death. In other embodiments, bacterial species of the present invention can be isolated by culturing a plant tissue extract or leaf surface wash in a medium with no added nitrogen source as described herein.
In one embodiment, the genomic DNA of the bacterial species shares least 97% and preferably at least 98% identity to SEQ ID NO:10. In some embodiments, the bacterial species is an Enterobacter species containing a set of genes required for nitrogen-fixation. In some embodiments, the Enterobacter species produces substantial amounts of extracellular polysaccharide (EPS), or endoglucanase. In another embodiment, the genomic DNA of the bacterial species shares 97% and preferably at least 98% identity to SEQ ID NO:11. In some embodiments, the bacterial species is a Pleomorphomonas species in which the nitrogen-fixing capability is stimulated by about 0.1 mM to about 0.5 mM of NH4+ ion. In an additional embodiment, the genomic DNA of the bacterial species shares 97% and preferably at least 98% identity homology to SEQ ID NO:12. In some embodiments, the bacterial species is a Sphingomonas species that is able to grow in nitrogen-free medium. In a further embodiment, the genomic DNA of the bacterial species shares 97% and preferably at least 98% identity to SEQ ID NO:13 or SEQID NO:14. In some embodiments, the bacterial species is a Methylobacterium species.
In a second aspect, the present invention provides an artificially formulated inoculant comprising at least one biologically pure culture of a bacterial species selected from the Enterobacter, Methylobacterium, Sphingomonas, and Pleomorphomonas genera as described herein. In one embodiment, the inoculant comprises a mixture of at least two biologically pure cultures of these bacterial species. In some embodiments, the preparation of the inoculant comprises large-scale production of the bacterial species described herein in a medium, optionally dehydrated in a suitable manner that maintains cell viability and the ability to artificially inoculate and colonize host plants. In some embodiments, the inoculant is optionally supplemented with trace metal ions. In one embodiment, the trace metal ions are selected from the group consisting of molybdenum (Mo2+) ions, iron (Fe2+) ions, manganese (Mn2+) ions, or any combination of these ions. In another embodiment, the concentration of the trace metal ions in the inoculant is between about 0.1 mM and about 50 mM. In some embodiments, the inoculant is formulated with a carrier. Any suitable carrier can be used and examples of suitable carriers include, but are not limited to, beta-glucan, carboxylmethyl cellulose (CMC), bacterial EPS, sugar and animal milk. In some embodiments, bacterial species that can be used for the inoculants of the present invention are those bacterial species described herein. In other embodiments, bacterial species that can be used for the inoculants of the present invention can be isolated by culturing a plant tissue extract or leaf surface wash in a medium with no added nitrogen source or sourced from a microbial collection centre based on taxonomy classifications and the characteristics of the bacterial species described herein. In some embodiments, the inoculant is effective in promoting plant growth, maintaining high chlorophyll content in leaves, increasing fruit or seed numbers, increasing fruit or seed unit weight and reducing NO2 emission due to reduced nitrogen fertilizer usage. In other embodiments, these properties are evidenced in plant species of the genera Jatropha, Sorghum, Gossypium, Elaeis, Oryza, Ricinus and Manihot. 
In a third aspect, the present invention provides a method for promoting plant productivity in a non-legume plant species. In some embodiments, the plant productivity is plant growth, maintenance of high chlorophyll content in leaves, increasing fruit or seed numbers, increasing fruit or seed unit weight or any combination of these characteristics. In other embodiments, plant productivity is also a reduction in NO2 emission which results from reduced nitrogen fertilizer use. In some embodiments, the non-legume plant species is selected from species of the genera Jatropha, Sorghum, Gossypium, Elaeis, Ricinus, Oryza and Manihot. In accordance with the present invention, the method comprises inoculating plants with a bacterial inoculant described herein. In one embodiment, the inoculant is sprayed on the plant aerial parts. In another embodiment, the inoculant is applied to the roots by inserting into furrows in which the plant seeds are planted, watering to the soil or dipping the roots in a suspension of the inoculant. In an additional embodiment, the inoculant is applied as a seed coating. In some embodiments, the inoculant comprises the trace metal ions described herein.
In other embodiments, the plant is sprayed with a trace metal ion solution that contains the trace metal ions described herein in addition to an inoculant which may not contain the trace metal ions.



BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1a and 1b show the effect of ammonium chloride addition on nitrogenase activity of Methylobacterium sp. strain. L2-4 (FIG. 1a) and Enterobacter sp. strain R4-368 (FIG. 1b). Nitrogenase activity was measured for cultures grown in 125-ml serum bottles containing N-free medium supplemented with different levels of ammonium chloride at 30° C. for 48 h. Error bars indicate ±SD.
FIG. 2 shows the effect of nitrogenase cofactor (Mo/Fe/Mn) addition on nitrogenase activity of Enterobacter sp. strain R4-368 (top) and Methylobacterium sp. strain L2-4 (bottom). Nitrogenase activity was measured for cultures grown in 125-ml serum bottles containing N-free medium supplemented with different levels of Mo/Fe/Mn at 30° C. for 48 h.
FIG. 3 shows the nitrogen-fixing activity in Jatropha of selected nitrogen-fixing strains in the presence of essential metals (Mo/Fe/Mn). Ethylene emission from Jatropha seedlings added with 15% acetylene (v/v) into the headspace was determined with and without inoculation of N-fixing root isolates. Acetylene reduction activity was calculated from the difference between inoculated with and without N-fixing root isolates. Values given are the means±standard deviations for triplicate determinations.
FIGS. 4a and 4b show the nitrogen-fixation gene cluster in Klebsiella pneumonia (Genbank No X13303) (FIG. 4a) and the homologous proteins as identified by BLASTP in Enterobacter sp R4-368 (SEQ ID NO:3) (FIG. 4b).
FIG. 5 shows the putative proteins encoded in SEQ ID NO:4 as identified by BLASTP.
FIGS. 6a and 6b show the nitrogenase activity of Pleomorphomonas jatrophae strain R5-392. FIG. 6a: Strain R5-392T grown in N-free medium and growth was recorded as OD600 nm at different time intervals. For measurement of nitrogenase activity, grown on N-free liquid cultures by injecting purified 15% acetylene (v/v) and incubate at 30° C. and then analyzed for ethylene production by GC. FIG. 6b: Nitrogenase switch-off by ammonium ions (top panel) and response to addition of nitrogenase cofactor Fe and Mo (bottom panel) in Pleomorphomonas jatrophae strain R5-392.



DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to novel clean technologies for agriculture and environment managements. In particular, the practice of this invention leads to reduced dependence on chemical nitrogen fertilizer; faster plant growth; improves crop productivity and reduced greenhouse gas NO2 emission in agriculture. In some embodiments, the present invention relates to methods of reducing chemical fertilizer usage and greenhouse gas nitrous oxide emission and to methods of improving plant growth rate and seed productivity in agriculture through the application of a novel artificially manufactured formula containing a nitrogen-fixing bacterium that efficiently colonizes non-legume plants in aerial parts and root system. The bacteria inocula and methods are particularly suitable for plants in the genera Jatropha, Sorghum, Gossypium, Elaeis, Otyza, Ricinus and Manihot. 
As used herein “bacterial inoculant,” “inoculant” or “inoculum” refers to a preparation that includes one or more bacterial species described herein.
The term “biologically pure culture” or “substantially pure culture” shall be deemed to include a culture of a bacterial species described herein containing no other bacterial species in quantities sufficient to interfere with the replication of the culture or be detected by normal bacteriological techniques.
The term “plant productivity” refers generally to any aspect of growth or development of a plant that is a reason for which the plant is grown. For example, when referring to food crops, such as grains or vegetables, crop productivity generally refers to the yield of grain or fruit, etc., harvested from a particular crop. Thus, for purposes of the present invention, improved plant productivity refers broadly to improvements in yield of grain, fruit, flowers, or other plant parts harvested for various purposes, improvements in growth of plant parts, including stems, leaves and roots, promotion of plant growth, maintenance of high chlorophyll content in leaves, increasing fruit or seed numbers, increasing fruit or seed unit weight, reducing NO2 emission due to reduced nitrogen fertilizer usage and similar improvements of the growth and development of plants.
In a first aspect, the present invention provides a biologically pure culture of a bacterial species selected from the Enterobacter, Methylobacterium, Sphingomonas, and Pleomorphomonas genera as described herein. In some embodiments, the bacterial species of the present invention are able to efficiently reduce atmospheric N2 to ammonia as evidenced by the AR assay in planta. In some embodiments, the N2 reduction occurs on the leaf surface. In other embodiments, the N2 reduction occurs inside the leaf surface. In additional embodiments, the N2 reduction occurs on the surface of the roots or root system. In further embodiments, the N2 reduction occurs inside the root tissue. In some embodiments, the bacterial species is able to self-propagate efficiently on the leaf surface, root surface or inside plant tissues without inducing a noticeable plant defense reaction, such as cell death. In other embodiments, bacterial species of the present invention can be isolated by culturing a plant tissue extract or leaf surface wash in a medium with no added nitrogen source, using suitable methods such as those described herein or similar methods well known to the skilled artisan or obvious modifications thereof.
In one embodiment, the genomic DNA of the bacterial species shares least 97% and preferably at least 98% identity to SEQ ID NO:10. In some embodiments, the bacterial species is an Enterobacter species containing a set of genes required for nitrogen-fixation. In some embodiments, the Enterobacter species produces substantial amounts of EPS, or endoglucanase. In another embodiment, the genomic DNA of the bacterial species shares 97% and preferably at least 98% identity to SEQ ID NO:11. In some embodiments, the bacterial species is a Pleomorphomonas species in which the nitrogen-fixing capability is stimulated by about 0.1 mM to about 0.5 mM of NH4+ ion. In an additional embodiment, the genomic DNA of the bacterial species shares 97% and preferably at least 98% identity homology to SEQ ID NO:12. In some embodiments, the bacterial species is a Sphingomonas species that is able to grow in nitrogen-free medium. In a further embodiment, the genomic DNA of the bacterial species shares 97% and preferably at least 98% identity to SEQ ID NO:13 or SEQID NO:14. In some embodiments, the bacterial species is a Methylobacterium species.
A biologically pure culture of Enterobacter species R4-368 and Pleomorphomonas jatrophae R5-392 were deposited on Jan. 6, 2012 under terms of the Budapest Treaty with the AGRICULTURAL RESEARCH SERVICE CULTURE COLLECTION (NRRL) (International Depositary Authority), National Center for Agricultural Utilization Research Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Ill. 61604 U.S.A., and assigned Accession Number NRRL B-50631 and NRRL B-50630, respectively. These strains can be cultured in R2A medium (Yeast extract 0.5 g/l; Proteose Peptone 0.5 g/l; Casamino acids 0.5 g/l; Glucose 0.5 g/l; Soluble starch 0.5 g/l; Na-pyruvate 0.3 g/l; K2HPO4 0.3 g/l; MgSO4*7H2O 0.05 g/l; agar 15 g/l, pH7.2).
A biologically pure culture of Methylobacterium sp. L2-4; Methylobacterium sp. L2-76 and Sphingomonas sp. S6-274 were deposited on Jan. 6, 2012 under terms of the Budapest Treaty with the AGRICULTURAL RESEARCH SERVICE CULTURE COLLECTION (NRRL) (International Depositary Authority), National Center for Agricultural Utilization Research Agricultural Research Service, U.S. Department of Agriculture 1815 North University Street, Peoria, Ill. 61604 U.S.A., and assigned Accession Number NRRL B-50628, NRRL B-50629 and NRRL B-50632, respectively. These strains can be cultured in 869 medium (Tryptone 10.0 g/l; Yeast extract 5.0 g/l; NaCl 5.0 g/l; D-glucose 1.0 g/l; CaCl2.2H2O 0.345 g/l; pH 7.0).
It is anticipated that certain mutants of the bacterial species described herein may also enhance plant growth comparable to the non-mutated forms. Mutants of the bacterial species described herein may include both naturally occurring and artificially induced mutants. Certain of these mutants will be found to useful using the assays described herein. Others mutants may be induced by subjecting the bacterial species described herein to known mutagens, such as N-methyl-nitrosoguanidine, using conventional methods. See, for example, U.S. Pat. No. 4,877,738 and U.S. Pat. No. 5,552,138, each incorporated herein by reference.
In a second aspect, the present invention provides an artificially formulated inoculant comprising at least one biologically pure culture of a bacterial species selected from the Enterobacter, Methylobacterium, Sphingomonas, and Pleomorphomonas genera as described herein. In one embodiment, the inoculant comprises a mixture of at least two biologically pure cultures of these bacterial species. In some embodiments, the preparation of the inoculant comprises large-scale production of the bacterial species described herein in a medium, optionally dehydrated in a suitable manner that maintains cell viability and the ability to artificially inoculate and colonize host plants. In some embodiments, the concentration of bacterial species in the inoculant is 108 to 1010 CFU/ml. In some embodiments, the inoculant is optionally supplemented with trace metal ions. In one embodiment, the trace metal ions are selected from the group consisting of molybdenum (Mo2+) ions, iron (Fe2+) ions, manganese (Mn2+) ions, or any combination of these ions. In another embodiment, the concentration of the trace metal ions in the inoculant is between about 0.1 mM and about 50 mM.
In some embodiments, the inoculant is formulated with a carrier. Any suitable carrier can be used. Examples of suitable carriers include, but are not limited to, beta-glucan, CMC, bacterial EPS, sugar and animal milk. Alternatively, peat or planting materials can be used as a carrier. In addition, biopolymers can be used as a carrier in which the inoculant is entrapped in the biopolymer. See, for example, U.S. Pat. Nos. 4,828,600, 5,061,490, 5,951,978 and No. 7,393,678, each incorporated herein by reference.
In some embodiments, bacterial species that can be used for the inoculants of the present invention are those bacterial species described herein. In other embodiments, bacterial species that can be used for the inoculants of the present invention can be isolated by culturing a plant tissue extract or leaf surface wash in a medium with no added nitrogen source or sourced from a microbial collection centre based on taxonomy classifications and the characteristics of the bacterial species described herein. In some embodiments, the inoculant is effective in promoting plant growth, maintaining high chlorophyll content in leaves, increasing fruit or seed numbers, increasing fruit or seed unit weight and reducing NO2 emission due to reduced nitrogen fertilizer usage. In other embodiments, these properties are evidenced in plant species of the genera Jatropha, Sorghum, Gossypium, Elaeis, Ricinus, Oryza and Manihot. 
In a third aspect, the present invention provides a method for promoting plant productivity in a non-legume plant species. In some embodiments, the plant productivity is plant growth, maintenance of high chlorophyll content in leaves, increasing fruit or seed numbers, increasing fruit or seed unit weight or any combination of these characteristics. In other embodiments, plant productivity is also a reduction in NO2 emission which results from reduced nitrogen fertilizer use. In some embodiments, the non-legume plant species is selected from species of the genera Jatropha, Sorghum, Gossypium, Elaeis, Ricinus, Oryza and Manihot. In accordance with the present invention, the method comprises inoculating plants with a bacterial inoculant described herein. In one embodiment, the inoculant is sprayed on the plant aerial parts. In another embodiment, the inoculant is applied to the roots by inserting into furrows in which the plant seeds are planted, watering the soil or dipping the roots in a suspension of the inoculant. In an additional embodiment, the inoculant is applied as a seed coating. In some embodiments, the inoculant comprises the trace metal ions described herein. In other embodiments, the plant is sprayed with a trace metal ion solution that contains the trace metal ions described herein in addition to an inoculant which may not contain the trace metal ions.
In one embodiment, the bacterial inoculant can be applied to plant 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 the skilled artisan. For example, the bacterial species described herein may be mixed with a porous, chemically inert granular carrier, such as described by U.S. Pat. No. 4,875,921, incorporated herein by reference. In another embodiment, the bacterial inoculant may be prepared with or without a carrier and sold as a separate inoculant to be used directly by the grower, such as by inserting directly into the furrows into which the seed is planted or by spraying the plant leaves. The process for inserting such inoculants directly into the furrows during seed planting is also generally well known to the skilled artisan, as is spraying the plants with the bacterial inoculant.
In general, the density of inoculation of the bacterial species onto seed, into furrows, or wetted on roots should be sufficient to populate the sub-soil region adjacent to the roots of the plant with viable bacterial growth. Similarly, the density of inoculation of the bacterial species sprayed onto plants should be sufficient to populate the leaves 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 plant productivity is improved.
It will be appreciated by the skilled artisan that a bacterial inoculant of the type described herein offers several significant potential advantages over the chemical inoculants or growth hormones or similar agents commonly used in agriculture today. By the very nature of the bacterial inoculant, the bacterial species are self-sustaining in a continuous fashion once the plants have been inoculated by any of the methods described herein. Therefore, there is no need for retreatment of the plants during the crop season. 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. Since the bacterial inoculant of the present invention can be sprayed onto plants, the application of this technique is relatively simple to the farmer. In these ways, a significant economic advantage is achievable.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Sambrook et al., 1989, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Ausubel et al., 1992), Current Protocols in Molecular Biology (John Wiley & Sons, including periodic updates); Glover, 1985, DNA Cloning (IRL Press, Oxford); Russell, 1984, Molecular biology of plants: a laboratory course manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Fire et al., RNA Interference Technology: From Basic Science to Drug Development, Cambridge University Press, Cambridge, 2005; Schepers, RNA Interference in Practice, Wiley-VCH, 2005; Engelke, RNA Interference (RNAi): The Nuts & Bolts of siRNA Technology, DNA Press, 2003; Gott, RNA Interference, Editing, and Modification: Methods and Protocols (Methods in Molecular Biology), Human Press, Totowa, N.J., 2004; Sohail, Gene Silencing by RNA Interference: Technology and Application, CRC, 2004.
EXAMPLES
The present invention is described by reference to the following Examples, which is offered by way of illustration and is not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below were utilized.
Example 1
Isolation of Diazotrophic Endophytes
Jatropha (Jatropha curcas L.) cultivars were sampled from the research plots of Agrotechnology Experimental Station located at Lim Chu Kang, Singapore. Plants were sampled from three trees of different cultivars that included origin from Indonesia, China and India. The root samples ( ̃1.2-2 cm dia) with adhering soil were carefully removed from root with a trowel and collected in sterile plastic bags. Stem cuttings ( ̃1.5 to 2.5 cm diameter) were removed from the plants with clippers and matured third leaves (non-pathogenic) from healthy branches that were placed on ice for shipment from the field to laboratory and processed immediately. Root, stem cuttings ( ̃50 g) and leaf ( ̃15-20 g) samples were thoroughly washed in distilled water, surface sterilized for 1 min in 90% ethanol followed by 10 min in 15% H2O2 and rinsed 3-5 times in sterile distilled water. A 100-μl sample of the water from the third rinse was plated on rich medium to verify the efficiency of sterilization. After sterilization, the roots and shoots were macerated in 10 ml 10 mM MgSO4 using a blender under sterile conditions. Serial dilutions were made, and 100-μl samples were plated on non-selective media in order to test for the presence of the endophytes and their characteristics. The bacteria were isolated from spread plate technique (100-μl) using different media with agar/phytagel such as N-free media (Nfb) (Baldani et al., 1980) for diazotrophs, rich media (M869) (Barac et al., 2004); for total heterotrophs and minimal medium (AMS) (Whittenbury et al., 1970); for methylotrophic bacteria. For isolation of diazotrophs, the suspension (10−to 10−5) were placed in duplicate tubes containing semi-solid N-free media and incubated for 96 h at 30° C. Growth pellicles in semi-solid media were streaked on to N-free agar media for single colony preservation and for further use.
Example 2
Bacterial Inoculants and Culture Conditions
Methylobacterium strains were grown in liquid medium 869 (Barac et al., 2004) with 1% methanol as the carbon source. Root isolates were cultivated in liquid medium 869 or 2xYT without methanol. All cultures were incubated at 30° C. and grown until early stationary phase. Just before inoculation, cells were harvested (10000 rpm, 8 min), washed once and resuspended in SDW or 10 mM MgSO4 solution, the optical density at 600 nm (OD600 nm) was adjusted, and inoculated as follows: for root inoculation, equal volume of cell suspension (OD600 nm=1.0 or 108 cfu ml−1) of each isolate was mixed and added at the time of sowing and at 15 days after sowing (DAS); for leaf inoculation, mixed or individual isolate was given as foliar spray till the wetting of leaves at 15 and 30 DAS (volume varied depending on the plant size and leaf number).
Example 3
nifH Amplification and Sequencing
The presence of the nifH gene was determined by a PCR as described by Pinto-Tomás et al. (2009). To amplify nifH gene using universal primers nif-Fo (5′-AAA GGY GGW ATC GGY AAR TCC ACC AC-3′; SEQ ID NO:1) and nif-Re (5′-TTG TTS GCS GCR TAC ATS GCC ATC AT-3′; SEQ ID NO:2) using the stringent PCR conditions described by Widmer et al. (1999) in order to reduce the emergence of falsely positive amplifications, including the following cycling conditions: 95° C./5min, 40 cycles of 94° C./15 s, 92° C./15 s, 54° C./8 s, 56° C./30 s, 74° C./10 s and 72° C./10 s, and final extension for 10 min/72° C. PCR products were eluted with QIAquick gel extraction kit prior to preparing sequencing reactions with the BigDye reaction mix and loaded into an Applied Biosystems 3700 automated DNA sequencing instrument. infH sequences from this study and close reference sequences obtained from the NCBI database by BLAST analysis.
Example 4
Nitrogenase Assays In Vitro
The acetylene-reduction (AR) assay was performed on free-living cultures as well as on cultures in association with crop plants. Nitrogen-fixing ability was determined by growing strains in 40 ml nitrogen-free medium (DSMZ medium no. 3) contained in a 125 ml serum bottle (Wheaton Industries Inc., USA). The medium contained the following (in 1 l distilled water): 5.0 g glucose, 5.0 g mannitol, 0.1 g CaCl2.2H2O, 0.1 g MgSO4.7H2O, 5.0 mg Na2MoO4.2H2O, 0.9 g K2HPO4, 0.1 g KH2PO4, 0.01 g FeSO4.7H2O, 5.0 g CaCO3 and 1 ml trace element mixture. The trace element mixture (SL-6, in DSMZ medium no. 27) contained the following (1−1 distilled water): 0.1 g ZnSO4.7H2O, 0.03 g MnCl2.4H2O, 0.3 g H3BO3, 0.2 g CoCl2.6H2O, 0.01 g CuCl2.2H2O and 0.02 g NiCl2.6H2O. Acetylene reduction was performed for all liquid cultures by injecting purified acetylene into appropriate containers sealed with gas-tight serum stoppers to yield 15% acetylene (v/v); this was followed by incubation for up to 96 h at 30° C. At intervals gas samples (0.5 ml) were removed by PTFE-syringe (Hewlett-Packard, USA) and analysed by Gas Chromatograph (GC 6890N, Agilent Technologies Inc., USA) with an flame ionization detector operated as follows: carrier gas, He; 35 ml/min; detector temperature, 200° C.; column, GS-Alumina (30 m×033 mm I.D.); pressure, 4.0 psi. Standard curve was prepared with ethylene (C2H4, Product Number: 00489, Sigma-Aldrich) injected in duplicate, the concentration ranged from 1 to 1000 n moles and calibrated (peak height vs nmol of C2H4). The protein concentration was determined by a modified Lowry method with bovine serum albumin as standard.
Example 5
Nitrogenase Assay in Planta
Acetylene-reduction (AR) assay was performed 2-3 weeks after inoculation, and the viable bacterial counts on shoot/roots were determined by serial dilution technique. Samples from each replication were collected from the glass house and most of the adhering soil was removed by shaking. Shoot/root/entire seedlings were inserted into the 125 ml glass bottles, closed with a 20 mm red stopper sleeve. After removing an equivalent volume of air, acetylene was injected into these bottles to give a final concentration of 15% and incubated at 30° C. for 4-12 h. Gas Chromatograph operating conditions was described by earlier. All values expressed were obtained after deducting the ethylene values for a blank treatment without samples.
Example 6
Enumeration of Bacterial Populations
Triplicate plant samples were randomly picked on each sampling, and each replicate consists of root and aerial parts from three plants. For all bacterial enumerations, the homogenates were serially diluted using sterile distilled water and plated on to AMS media with 0.5% methanol to determine the Methylobacterium population or to M869 plates for total bacterial population. Bacterial colonies were counted after incubating the plates for 5 days at 30° C.
Example 7
In Vitro and in Planta AR Activity of Selected Diazotrophic Isolates
Many diazotrophic species were isolated from jatropha plant or rhizophere. The nitrogen-fixing capability was confirmed by the presence of nifH-like gene by PCR, AR activity in vitro and in planta. We found that nitrogenase activity in isolate bacteria culture is significantly different from their performance observed in planta. Table 1 lists the activity of selected strains.







TABLE 1







AR Activity of Selected Diazotrophic Isolates














Nitrogenase







activity in pure
In planta AR




culture (nmol
activity (nmol




C2H4 h− 1 mg
C2H4 h−1
nifH
Endoglucanase


Selected strains
Medium used
protein−1)
plant−1)
gene*
Activity





Methylobacterium sp.
JNFb
661.3 ± 65.31
94.17 ± 9.52
+(420 nt)
+


L2-4


Methylobacterium sp.
JNFb
272.2 ± 20.15
32.70 ± 5.21
+
+


L2-76


Methylobacterium sp.
JNFb
223.3 ± 32.14
61.57 ± 7.62
+
+


L7-515


Enterobacter sp0 R4-
DSMZ**
1060.7 ± 110.6 
220.08 ± 20.08
+(432 nt)
+


368


Enterobacter sp. R5-
DSMZ
960.5 ± 110.5
77.54 ± 8.54
+
+


362


Enterobacter sp. R4-
DSMZ
1012.8 ± 62.8 
 71.13 ± 10.07
+
+


390


Pleomorphomonas
DSMZ
92.79 ± 4.8 
Nd
+(409 nt)
−


jatrophae R5-392


Sphingomonas sp. S6-
DSMZ
16.14 ± 1.2 
Nd
+
+


274





Note:


*nifH gene - amplified using PCR and the products are sequenced for further confirmation.


**DSMZ N-free medium no. 3; Acetylene reduction was performed for all liquid cultures by injecting purified acetylene into appropriate containers sealed with gastight serum stoppers to yield 15% acetylene (v/v); this was followed by incubation for up to 96 h at 30° C. Ethylene peak is not detected in control bottle without addition of 15% acetylene (v/v) into the headspace. Each value is the mean ± SD of three replications per treatment.


Nd: not determined.






Example 8
Effect of Ammonium Ion on Nitrogen-Fixing Activity
Resistance to ammonia is a critical feature of nitrogen-fixing bacteria inoculants for this invention. To demonstrate this, we assay the AR activity of two best candidates of our inoculants under the conditions with various concentration of NH4Cl. Surprisingly, both Enterobacter sp. R4-368 and Methylobacterium sp. L2-4 showed not only strong activity up to 1 mM but also significantly enhanced activity by the NR4+ ion although inhibitory effect was observed at about 5 mM (FIGS. 1a and 1b).
Example 9
Effect of Metal Ions on N-Fixation
We assayed nitrogenase activity in media supplemented Na2MoO4 or FeSO47H2O at various concentrations (0, 0.1, 0.5, 1, 10 mM) in N-free medium. Nitrogenase activity was clearly stimulated by Fe2+ and molybdenum (Mo2+) ions (FIGS. 2 and 3).
Example 10
Agronomical Performance of Jatropha curcas Inoculated with N-Fixing Strains
To confirm the efficacy of nitrogen-fixing bacterial inoculants in plants, selected strains were inoculated to Jatropha curcas seedlings either in a single isolates or mix isolates. Plants were grown in the open air in plastic pots (30 cm in diameter) after bacterial inoculation. From the time of flowering to harvesting, commercial NPK fertilizers was applied at half of the recommended dose (0.5 g/plant) once in 15 days intervals. As shown in Table 4, significant improvements in chlorophyll content, leaf AR activity, seed yield and single seed weight were observed. Noticeably, mixed inoculation of root and leaf isolates lead to best chlorophyll content and AR activity. Flowering time was also shortened, particularly in treatments with L2-4 strains. The relatively high AR activity in control plants may result from naturally occurring N-fixing species from the environment. Importantly, total seed yields were increased to about 4-fold when inoculated with a single leaf isolate L2-4 or mix root isolates (Table 2).







TABLE 2







Effect of N-Fixing Strains on Jatropha curcas
















AR-
Average


Total seed




Relative
activity
time to
No. of

weight per



Chlorophyll
(nmol C2H4
flowering
fruits per
Number of
treatment
Single seed


Treatments
content
h−1 g leaf1)
(DAP)
treatment
seeds/plant
(g)
weight (g)

















Control
33.29 ± 2.44
18.11 ± 2.1
137
26 ± 2.87
75 ± 8.14
36.25
0.483


L2-4
38.17 ± 4.10
22.31 ± 3.3
110
101 ± 7.91 
261 ± 20.78
134.89
0.517


L2-76
36.47 ± 4.01
20.95 ± 4.0
125
58 ± 4.37
157 ± 11.41
81.56
0.519


Leaf isolates
37.66 ± 4.17
24.00 ± 4.0
118
43 ± 2.72
119 ± 8.13 
62.63
0.526


(LI)


Root isolates
38.12 ± 3.30
19.97 ± 2.0
124
98 ± 9.00
286 ± 25.47
145.27
0.508


(RI)


RI + LI
40.07 ± 5.14
36.67 ± 5.7
115
73 ± 5.03
182 ± 12.41
92.05
0.506


(MI)





Note:


Each value is the mean ± SD of eight replications (n = 8). Leaf isolates (LI): L2-4 (Methylobacterium sp.), L2-76 (Methylobacterium sp.) and S6-274 (Sphingomonas sp.); Root isolates (RI): R4-368 (Enterobacter sp.), R5-362 (Enterobacter sp.), R5-431 (Klebsiella sp.), R7-601 (Sinorhizobium sp.) and R1-99 (Rhizobium sp.). For treatments that include leaf isolates, the cultures were applied using a hand sprayer (25 ml per pot; 1 × 109 CPU ml−1 of culture or wetting of all the leaves) at 30 and 90 days after planting (DAP). Chlorophyll content and AR-activity measured on 30 days after inoculation (DAI). Mature fruits were collected continuously and dried at 37° C. for 2 days. Seeds were collected from dried fruits and stored in 4° C. for further analysis. Fruit and seed yield data was recorded over a period of 7 months.






To demonstrate that the bacteria species are able to self-propagate in plant tissues, non-surface sterilized homogenates were serially diluted using sterile distilled water and MIN techniques were performed with 3 tubes containing Nfb media using from 10−3 to 10−5 dilutions and incubated at 30° C. for 7 days. For endophytic population before homogenization, leaf and root tissues were surface-sterilized by immersion in 70% ethanol, 1 min; 15% H2O2 for 1 min followed by 5-10 thorough rinsing in sterile distilled water. Serial dilutions were also plated on agar N-free media. Bacterial colonies were counted after incubating the plates for 5 days at 30° C. Results are shown in Table 3. For heterotrophic and methlotrophic population were showed the same trend as higher population of leaf associated bacteria than endophytic populations (data not shown).







TABLE 3







Diazotrophic Bacterial Populations (MPN Techniques)









Population (Log cfu/g sample)









Treat-
Leaf sample
Root sample











ments
Leaf associated
Endophytic
Root associated
Endophytic





Control
3.97 ± 0.97
2.48 ± 0.18
2.56 ± 0.16
2.46 ± 0.36


L2-4
5.38 ± 0.38
2.96 ± 0.46
3.51 ± 0.41
2.81 ± 0.11


L2-76
5.61 ± 0.51
2.82 ± 0.72
3.16 ± 0.36
2.64 ± 0.34


Leaf
5.46 ± 0.26
3.10 ± 0.30
3.24 ± 0.54
2.59 ± 0.19


isolates


(LI)


Root
4.12 ± 0.12
3.05 ± 0.25
4.32 ± 0.32
3.65 ± 0.25


isolates


(RI)


RI + LI
5.72 ± 0.62
3.21 ± 0.31
4.66 ± 0.66
3.88 ± 0.43


(MI)





Note:


Leaf associated or endophytic populations from both surface and non-surface sterilized leaves were measured in Nfb media at the time of fruit harvesting stage i.e. 6 months after 2nd inoculations.






Example 11
Promotion of Jatropha Seedling Growth
Jatropha (cv. MD44) seeds were surface sterilized by immersion in 70% ethanol, 1 min; 2% NaOCl for 30 sec followed by 5-10 thorough rinsing in sterile distilled water. For plant inoculation experiments, Methylobacterium strains (leaf isolates, L2-4 and L7-515) were grown in liquid 2xYT medium (root isolates R4-368, R5-362, R5-431, R7-601 and R1-99 were grown in without methanol) containing methanol (30 mM) to midexponential growth phase, centrifuged, washed, resuspended in sterile distilled water and stored in 4° C. until use. The bacteria were adjusted to an OD600 nm of 1.2 (108 cfu per ml) and used for plant inoculation after sterilization of Jatropha seeds. For mixed inoculation, equal volumes of five cultures were mixed and then used for seed imbibition (in RT at 6 h for 60 rpm), root application (2 ml per pot after sowing) and phyllosphere spray till wetting of leaves.
Under glass house growth conditions, the analysis that was carried out at 45 DAS showed that for most of the plant parameters examined, there was a significant increase in N-fixing bacteria-treated plants compared to non-inoculated control plants (Table 4). In Jatropha, individual inoculation of N-fixer (LI and RI) or its mixed-inoculation produced significant increases in number of leaves, chlorophyll content, seedling vigour, rate of germination and biomass compared to control plants or plants individually-inoculated with LI or RI. Under greenhouse condition, the AR activity in the control plants are much lower, a likely consequence of less cross-contamination.







TABLE 4







Seedling Vigour Index, Number of Leaves and Germination


Rate of N-Fixer-Treated Jatropha seeds (45 DAS)














No. of
Relative
Nitrogenase


Biomass


Treatments†
Leaves
Chlorophyll
activity‡
SVI
RG
(g)





Control
6.19 ± 0.29
34.91 ± 3.91
 4.15 ± 0.25
901.7 ± 46.7
23.50 ± 2.5 
46.12 ± 6.12


Root isolates
6.58 ± 0.48
36.08 ± 4.08
 8.56 ± 1.06
1054.1 ± 60.18
24.55 ± 2.55
62.0 ± 4.0


(RI)


Leaf isolates
6.47 ± 0.42
38.81 ± 4.31
16.79 ± 2.79
 986.2 ± 65.22
23.57 ± 2.57
61.4 ± 3.4


(LI)


RI + LI
6.72 ± 0.42
35.31 ± 3.81
11.52 ± 1.72
1037.6 ± 67.51
25.41 ± 2.41
63.0 ± 4.0





Note:


†Average of 150 Jatropha seedlings.


‡Value expressed in nmol C2H4 day−1 seedlings−1. Each value represents the means of four replicates per treatment. Seedling vigour index (SVI) was calculated using the formula: SVI = % germination × seedling length (shoot length + root length) in cm (Baki and Anderson, 1973). Rate of germination (RG) was calculated using the following formula, RG = Σ Ni/Di where Ni is the number of germinated seeds in a given time, and Di is the time unit (day) (Madhaiyan et al., 2004).






The plant growth promoting effect can be observed in extended period in Ø35 cm pots. Significant improvements in plant height, stem diameter, leaf number and stem volume were observed (Table 5). Soil with poor nutrient content was used and a commercial NPK fertilizer was applied at half of the recommended dose (0.5 g/plant) once in 15 days intervals.







TABLE 5







Combined Inoculation of N-Fixer on the


Early Growth of Jatropha (90 DAS)
















Relative




Plant height
Stem
No. of
chlorophyll
Stem


Treatments
(cm)
Diameter
leaves
content
Volume





Control
32.23
25.55
11.58
26.20
167.41


RI
40.37
27.72
20.58
33.40
244.08


RI + LI
43.97
27.44
24.50
38.16
261.59





Note:


Each value is the mean of 12 plants per treatment (n = 12).


Leaf isolates (LI): L2-4 (Methylobacterium sp.) and L7-515 (Methylobacterium sp.);


Root isolates (RI): R4-368 (Enterobacter sp.), R5-362 (Enterobacter sp.), R5-431 (Klebsiella sp.), R7-601 (Sinorhizobium sp.) and R1-99 (Rhizobium sp.).






Example 12
Effects of Methylobacterim sp Inoculation in Cotton, Castor Bean, and Sorghum 
Seeds were surface sterilized by immersion in 70% ethanol, 1 min; 2% NaOCl for 30 sec followed by 5-10 rinsing in sterile distilled water. For plant inoculation experiments, Methylobacterium strain L2-4 was grown in liquid 2xYT medium containing 30 mM methanol (v/v) to mid-exponential growth phase, centrifuged, washed, resuspended in sterile distilled water and stored in 4° C. until use. For seed imbibitions, the surface sterilized seeds were soaked in a double volume of bacterial culture (OD600 nm=1.2 or 109 cfu ml−1 of culture). After 6 h soaking, the culture was drained, and the seeds were dried in shade for 30 min before being allowed to sprout for another 24 h prior to sowing. For phyllosphere spray, the cultures were applied using a hand sprayer (approximately 25 ml per plant; OD600 nm=1.2) at 15 DAS.
Under greenhouse conditions, Methylobacterium strain L2-4 showed good AR activity in leaves of cotton, castor, sorghum, and rice (Table 6). The effects on crop growth are shown in Table 7.







TABLE 6







Nitrogenase (or Acetylene-Reduction) Activity of Various


Crops Inoculated with Methylobacterium sp. strain L2-4 (20 DAI)










AR-activity (nmol C2H4 d−1
AR-activity (nmol C2H4 d−1


Crops
seedlings−1)
g−1 sample (DW)





Cotton
176.67 ± 21.67
 70.7 ± 11.72


Sorghum
259.61 ± 59.61
177.8 ± 28.77


Castor bean
25.78 ± 5.78
19.2 ± 5.20


Rice
17.54 ± 2.54
140.8 ± 15.77


Jatropha
288.45 ± 78.45
114.5 ± 19.46


(MD-44)





Note:


Each value is the mean ± SD of three replications per treatment.













TABLE 7







Effect of Inoculation of Methylobacterium sp. L2-4 on Crop Growth













Plant height
No. of leaves
Relative chlorophyll
No. of branches
Number flower



(cm)
(#)
content*
(#)
(#)

















Crops
Control
Treated
Control
Treated
Control
Treated
Control
Treated
Control
Treated





Cotton
101.5 ±
103.4 ±
40 ±
44.25 ±
35.95 ±
39.195 ±
5.75 ±
8.75 ±
9.25 ±
15.75 ±



4.45
3.08
2.16
1.89
1.12
1.89
0.50
0.96
1.5
1.26


Castor
105.8 ±
115.8 ±
10 ±
12.25 ±
49.95 ±
54.575 ±
ND
ND
0
2 (4)


bean
12.21
3.92
2.16
1.50
4.28
1.97


Sorghum
146.5 ±
138.3 ±
17 ±
19.25 ±
51.08 ±
61.15 ±
ND
ND
1.5 ±
1.5 ±



31.41
23.45
2.16
4.99
4.04
3.61


1.0
0.58


Jatropha
70.8 ±
72.0 ±
16.75 ±
20.75 ±
30.66 ±
34.18 ±
ND
ND
ND
ND



1.71
2.45
0.5
4.50
0.97
1.62





Note:


Each value is the mean ± SD of four replications per treatment. Relative chlorophyll content was measured 5 leaves from top of the plants. Results were recorded at 75 days after inoculation.






Example 13
AR Activity and Taxonomy Classification data of Pleomorphomonas jatrophae 
The N-fixation ability could be proved by a multidisciplinary approach. Strain R5-392T was subjected to a nifH-specific PCR amplification. The expected 409 by amplification product was observed with strain R5-392 tested. In the case of strain R5-392, this PCR product was purified and sequenced. The comparison of the resulting sequence with the EMBL database revealed a similarity with the nifH gene of P. oryzae of 98%. The resulting amino acid (130 deduced amino acids) sequence had 100% similarity to the ATP-dependent reductase or nitrogenase iron-protein (nitrogenase component II, dinitrogenase reductase, nifH protein) of P. oryzae. The nitrogenase activity of the strain R5-392 was tested by the acetylene-reduction assay method. Strain R5-392T grown in N-free medium, effectively reduced acetylene and exhibited highest nitrogenase activity of >300 nmol C2H4 ml−1 in the headspace gas sample after 96 h incubation at 30° C. with the population increase of >6 log CFU ml−1 (FIG. 6a). However, close relatives of R5-392 showed less than 10 nmol C2H4 produced per mg of protein (data not shown). Lower concentrations of nitrogen (0.1 mM NH4Cl) in N-free medium have triggered higher nitrogenase activity than higher concentrations or no NH4Cl in the medium (FIG. 6b). Nitrogenase system was switched-off using NH4Cl at concentrations >1 mM in the N-free medium. Nitrogenase activity was measured by supplementing Na2MoO4/FeSO4.7H2O at various concentrations (0, 0.1, 0.5, 1, 10 mM) in N-free medium and injected with 15% acetylene (v/v) after inoculation. Nitrogenase activity was higher in 0.1 mM Fe concentration and reduced with increasing concentrations of Fe. No nitrogenase activity was recorded in N-free medium without Fe (FIG. 6b).
Example 14
Genome Analysis for Enterobacter sp R4-369 and Methylobacterium sp. L2-4
Total DNA was extracted for Enterobacter sp R4-369 and Methylobacterium sp. L2-4 and a partial genomic sequence was obtained for short-gun sequencing by the 454 pyrosequencing. Using the Klebsiella pneumonia nif gene cluster as a query (Genbank No 13303), a full set of nif genes were identified (FIG. 4). The sequence for Methylobacterium sp. L2-4 is set forth in SEQ ID NO:3. In contrast, no such genes can be identified in either Enterobacter cloacae (GenBank Accession Nos. NC_014121.1 and NC_014618.1) nor the endophytic plant growth-promoting Enterobacter sp. 638 (GenBank Accession No. NC_009436.1).
Surprisingly, no clear homologue was found in the genome of Methylobacterium sp. L2-4 nor the published Methylobacterium radiotolerans JCM 2831 genome (GenBank Accession Nos. CP001001-CP001009), which share about 99% sequence identity in 16S rDNA and total sequence. A Blast search using the K pneumonia nifH protein sequence as query identified distantly related proteins, GenBank Accesssion Nos. YP_001755485.1 and YP_001754523.1 that share about 35% amino acid identity and was predicted to encode the chlorophyllide reductase iron protein subunit X and protochlorophyllide reductase iron-sulfur ATP-binding protein respectively. This is much lower than that between the K. pneumonia nifH and cyanabacteria nifH (GenBank Accession No.ACC79194.1). The genome also lack obvious nif gene cluster. SEQ ID NO:7 encode a putative nifH orthologue. Interestingly, the neighboring putative coding sequences for protochlophyllide reductase subunits share significant homology to nifD and nifE (FIG. 5). As L2-4 is able to fix nitrogen, it is probable that these genes are critical for nitrogen-fixation in Methylobacterium. 
Example 15
Effect of EPS Production on N-Fixation in Enterobacter 
N-fixing defective mutants were identified by Tn5 mutagenesis. EPS mutants were identified as small non-motile colonies on N-free solid medium. All EPS mutants have negligible nitrogenase activity as determined by AR assay. Some of the mutants are summarized in Table 8.







TABLE 8







EPS Mutants









Mutant




ID
Gene inserted
Tn5 flanking sequence (SEQ ID NO:)





S238
Colanic acid polymerase
cgttaatctcaatttggctcagcatcaaacagtttggtat (5)


 


S236
EPS exporter
gcgttggcccaggggatgtgctgaacgtaaccgtg (6)


 


S210
Colanic acid
ccccgcaggtgctgtatcgctggcgcgcatt (7)



acetyltransferase WcaF



 


S237
GDP-L-fucose synthetase
ctggtgctgcgttcccgcgaggagctgaacc (8)


 


S300
Colanic acid biosynthesis
cgtctgaacgttgaatcgcggcacgacgagga (9)



UDP-glucose lipid carrier




transferase WcaJ









Example 16
Genome Sequencing and 16S rRNA Gene Amplification and Sequencing
Genomic DNA extractions were carried out according to standard protocol (Wilson, 1997) and purified using DNeasy mini spin column (Qiagen Cat. No.: 69106) and sequenced using Genome Sequencer FLX Titanium (GS-FLX Titanium) technology at Macrogen (Republic of Korea). Shotgun sequencing and mate-pair end sequencing (3 kb) were performed and sequence assembly of quality filtered reads was performed using a GS De Novo assembler (v 2.6).
16S rRNA gene was amplified using universal primers 27F and 1492R were used (Delong, 1992). Cycling conditions were as follows: initial denaturation for 10 min at 95° C.; then 30 cycles of 1.5 min at 95° C., 1.5 min at 55° C. and 1.5 min at 72° C.; and a final extension for 10 min at 72° C. PCR products were ligated into a plasmid vector pGEM-T Easy (Promega, Madison, USA) and transformed into competent cells of Escherichia coli strain XL-Blue by using a pGEM-T Easy Vector System (Promega). After the transformants were cultured overnight at 37° C. on Luria-Bertani (LB) agar plates containing ampicillin (100 μg ml−1), positive transformants were cultured overnight on LB liquid medium. Plasmids were prepared by using QIAprep Miniprep (Qiagen). Sequences of the 16S rRNA genes were obtained by using primers 27F (5′-AGAGTTTGATCMTGGCTC-3′; SEQ ID NO:15), 1492R (5′-TACCTTGTTACGACTT-3′; SEQ ID NO:16), 785F (5′-GGATTAGATACCCTGGTA-3′; SEQ ID NO:17), 518R (5′-GTATTACCGCGGCTGCTGG-3′; SEQ ID NO:18), 1100R (5′-GGGTTCGCTCGTTG-3′; SEQ ID NO:19) and their nucleotide sequences were determined with an Applied Biosystems 3730 XI DNA sequencer (AB Applied Biosystems, HITACHI). The 16S rDNA sequences were aligned by using the Megalign program of DNASTAR. Sequence similarity analyzed by the EzTaxon server (http://www.eztaxon.org/) (Chun et al., 2007).
Example 17
Effect of Inoculation of Enterobacter and Methylobacterium Sp on Oil Palm
Seedling of micropropagated interspecies oil palm hybrid clones which were potted in non-sterilized soil were inoculated by pouring into the soil (for enterobacter R4-368) or sprayed until dripping to the leaves (for Methylobacterium sp L2-4). Plants were grown in the open air. Compared with mocked inoculated plants, plant height was significantly improved when inoculated both strains. For leaf number, all treatment gave a significantly higher leaf numbers (Table 8). These results were associated with a higher endophytic nitrogen fixing bacteria counts in the root and on the upper leaf surface (Table 10).







TABLE 9







Plant Height and Leave Number at 150 Days After Inoculation



















Plant series No.
P1
P2
P3
P4
P5
P6
P7
P8
P9
Avr
STDEV
P value





Plant Height














Mock inoculated
60
55
56
35
61
54
59
60
55
55.00
7.94


L2-4
42
55
52
69
53
68
57
67
51
57.11
9.16


R4-368
57
54
58
65
61
61
60
69
59
60.44
4.42
0.09


L2-4 + R4-368
56
63
66
68
48
61
69
65
71
63.00
7.21
0.04


Leave Number


Mock inoculated
10
10
 9
11
10
10
11
10
10
10.11
0.60


L2-4
12
13
13
13
13
12
12
13
13
12.67
0.50
0.0005


R4-368
11
11
12
12
12
12
12
11
11
11.56
0.53
0.0005


L2-4 + R4-368
13
13
11
12
12
12
12
11
11
11.89
0.78
0.0005
















TABLE 10







Nitrogen Fixing Bacteria Counts at 150 Days After Inoculation










Root




population1
leaf population2











Treatments
cfu per g
STDEV
cfu/cm2
STDEV














Mock inoculated
1.27 × 10{circumflex over ( )}6
0.20
0
0


L2-4
Nd

7.33
0.88


R4-368
3.68 × 10{circumflex over ( )}6
0.05
Nd


L2-4 + R4-368
1.85 × 10{circumflex over ( )}7
0.22
8.00
1.73





Note:


1root were surface-sterlized before glinding.


2Non-sterlized upper leaf surface were imprinted on agar plates and the red-pigmented colonies were scored 3 days after incubation in 30° C.






Example 18
Effect of Nitrogen-Fixation Genes on Growth Promotion and Leaf N Content
Knockout mutant of nifH, nifD and nifK genes were created in Enterobacter R4-368 by electroporation of the respective knockout construct containing about 1 kb flanking sequences fused at both sides of a synthetic mini-Tn5 transposon (Kanamycin resistant). Clean knockout mutants were confirmed by PCR and Southern blotting. The mutant and wildtype strains were inoculated to the root system of plants derived from surface sterilized Jatropha curcas seeds and potted in sterilized sand and vermiculite (1:1) mix. Inoculation was done by pouring 100 ml cell preparation (1 OD600 in water) into each pot. Plants were grown in open air in a greenhouse. Plant height, number of leaves and dry biomass were measured at 45 days after inoculation. Dried leaf biomass ( ̃2 mg) from 4 plants was also subjected to nitrogen content analysis, which was done with the Elementar Vario Micro Cube (ELEMENTAR Analysensysteme, Germany). As can be seen in Table 11, all knockout mutants showed similar plant height, leaf number, biomass and N content to mock inoculated plants whereas significant improvements were observed in plants inoculated with wildtype Enterobacter sp. R4-368.







TABLE 11







Effect of nifHDK Gene Deletion in Enterobacter sp. R4-368 at 45 Days After Inoculation














No. of
Shoot
Root
Leaf N content


strains
Plant height (cm)*
leaves*
biomass (g)*
Biomass (g)*
(%)#





Wildtype
34.0 ± 5.8
10.0 ± 2.4 
7.1 ± 0.7
1.4 ± 0.1
1.968 ± 0.323


nifHΔ
25.3 ± 4.5
8.1 ± 1.2
4.1 ± 1.3
0.7 ± 0.1
1.055 ± 0.013


nifDΔ
23.1 ± 4.5
7.1 ± 1.2
4.4 ± 0.4
0.6 ± 0.1
0.878 ± 0.019


nifKΔ
22.6 ± 3.9
7.3 ± 1.3
4.4 ± 0.5
0.8 ± 0.1
1.263 ± 0.025


mock
17.9 ± 1.3
6.1 ± 1.2
2.6 ± 0.5
0.6 ± 0.0
0.903 ± 0.025





*n = 7;


#n = 4






Example 19
Effect of EPS Gene Cluster on Plant Colonization
Jatropha seed (cultivar MD-44) coats were removed, seed kernel were sterilized with 75% ethanol (v/v) for 1 min followed by 10% H2O2 (v/v) for 60 min followed by five subsequent rinses in sterile distilled water (SDW) and immersed in SDW overnight at 28° C. in darkness. The endosperm-free embryos were germinated on hormone-free seed germination medium (1⁄2 MS salt, B5 Vitamins, 5 g/L sucrose, 0.5 g/L MES and 2.2 g/L phytagel, pH 5.6) and cultured in a tissue culture room, at 25° C.±2° C. in a 16 h light (100 μηiol/iη28)/8 h dark cycle. Strains used are wild-type Enterobacter sp. R4-368 , Tn5 mutant S300 which contains a Tn5 transposon in the EPS gene cluster and produce very little EPS and the knockout mutant that has the whole EPS gene cluster deleted. The mutant and wildtype strains were inoculated to the root system of plants derived from surface sterilized Jatropha curcas seeds and potted in sterilized sand and vermiculite (1:1) mix. Inoculation was done by pouring 100 ml cell preparation (1 OD600 in water) into each pot. Plants were grown in open air in a greenhouse with regular watering of water or nutrient solutions. Both the Tn5 mutant and knockout mutant of the EPS genes showed significant reduction (p<0.05) in plant heights and biomass (Table 12).







TABLE 12







Effect of Enterobacter or EPS Genes on Jatropha Growth Promotion










Plant height
Dry biomass (g)2










Strains
(cm)1
Shoot
Root





Enterobacter sp. R4-368
28.1 ± 2.90
9.50 ± 0.57
1.74 ± 0.04


Knockout EPS 130-1
24.9 ± 2.90
6.75 ± 0.76
1.15 ± 0.17


sTn5 mutant S300
26.0 ± 1.50
6.90 ± 0.31
1.14 ± 0.12


Mock innoculated
23.3 ± 2.30
7.57 ± 0.30
0.96 ± 0.20





Note:


Each value represents the mean and SD of 7 plants.


130 days after inoculation.


245 days after inoculation.






The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10-15 is disclosed, then 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the pracfice of the invention.
It will be appreciated that the methods and compositions of the instant invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
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## Claims

1. A bacterial inoculant for application to plants, said bacterial inoculant comprising an effective quantity of a biologically pure culture of at least one bacterial species, trace metal ions and a carrier;
wherein the trace metal ions are selected from the group consisting of molybdenum (Mo2+) ions, iron (Fe2+) ions, manganese (Mn2+) ions, and any combination of these ions;
wherein the carrier is selected from the group consisting of a biopolymer, a sugar, an animal milk, a planting material and a porous, chemically inert carrier;
wherein the at least one bacterial species is Enterobacter species R4-368 (NRRL B-50631); and
wherein the bacterial species reduces acetylene to ethylene or reduces atmospheric nitrogen (N2) to ammonia (NH3) and improves plant productivity when the bacterial inoculant is applied to a plant.

2. The bacterial inoculant of claim 1, wherein the reduction occurs on the leaf surface, inside the leaf surface, inside the root tissue or on the surface of the root system.

3. The bacterial inoculant of claim 1, wherein the reduction occurs in association with a plant selected from the group of plant species of a Jatropha genus, Sorghum genus, Gossypium genus, Elaeis genus, Ricinus genus, Oryza genus and Manihot genus.

4. The bacterial inoculant of claim 1, further comprising a bacterial species selected from the group consisting of Pleomorphomonas jatrophae R5-392 (NRRL B-50630), Methylobacterium sp. L2-4 (NRRL B-50628), Methylobacterium sp. L2-76 (NRRL B-50629), Sphingomonas sp. S6-274 (NRRL B-50632) and any combination thereof.

5. The bacterial inoculant of claim 1, wherein the bacterial species secrets exopolysaccharide (EPS) or endoglucanase.

6. The bacterial inoculant of claim 1, wherein the concentration of the trace metal ions in the inoculant is between about 0.1 mM and about 50 mM.

7. A method of improving plant productivity comprising inoculating a plant with an effective quantity of the bacterial inoculant of claim 1.

8. The method of claim 7, wherein the bacterial inoculant is sprayed on aerial plant parts.

9. The method of claim 7, wherein the bacterial inoculant is applied by inserting into a planting furrow, watering the plant root system in the soil, dipping the roots in the bacteria inoculant or coating seeds.

10. The method of claim 7, wherein the concentration of the trace metal ions in the inoculant is between about 0.1 mM and about 50 mM.

11. The method of claim 10, wherein the bacterial inoculant is sprayed on aerial plant parts.

12. The method of claim 7, wherein the plant productivity is plant growth, maintenance of high chlorophyll content in leaves, increasing fruit or seed numbers, increasing fruit or seed unit weight or any combination of these characteristics.

13. The method of claim 7, wherein the plant is a species of a Jatropha genus, Sorghum genus, Gossypium genus, Elaeis genus, Ricinus genus, Oryza genus or Manihot genus.

14. The method of claim 10, wherein the plant productivity is plant growth, maintenance of high chlorophyll content in leaves, increasing fruit or seed numbers, increasing fruit or seed unit weight or any combination of these characteristics.

15. The method of claim 10, wherein the plant is a species of a Jatropha genus, Sorghum genus, Gossypium genus, Elaeis genus, Ricinus genus, Oryza genus or Manihot genus.

16. A method of improving plant productivity comprising inoculating a plant with an effective quantity of the bacterial inoculant of claim 4.

17. The method of claim 16, wherein the plant productivity is plant growth, maintenance of high chlorophyll content in leaves, increasing fruit or seed numbers, increasing fruit or seed unit weight or any combination of these characteristics.

18. The method of claim 16, wherein the plant is a species of a Jatropha genus, Sorghum genus, Gossypium genus, Elaeis genus, Ricinus genus, Oryza genus or Manihot genus.

19. The bacterial inoculant of claim 4, wherein the concentration of the trace metal ions in the inoculant is between about 0.1 mM and about 50 mM.

20. The method of claim 16, wherein the bacterial inoculant is sprayed on aerial plant parts.

21. The method of claim 16, wherein the bacterial inoculant is applied by inserting into a planting furrow, watering the plant root system in the soil, dipping the roots in the bacteria inoculant or coating seeds.

22. The method of claim 16, wherein the concentration of the trace metal ions in the inoculant is between about 0.1 mM and about 50 mM.

23. The method of claim 22, wherein the bacterial inoculant is sprayed on aerial plant parts.

24. The method of claim 22, wherein the plant productivity is plant growth, maintenance of high chlorophyll content in leaves, increasing fruit or seed numbers, increasing fruit or seed unit weight or any combination of these characteristics.

25. The method of claim 22, wherein the plant is a species of a Jatropha genus, Sorghum genus, Gossypium genus, Elaeis genus, Ricinus genus, Oryza genus or Manihot genus.

26. The method of claim 22, wherein the bacterial inoculant is applied by inserting into a planting furrow, watering the plant root system in the soil, dipping the roots in the bacteria inoculant or coating seeds.