US 2013 O150240A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0150240 A1 Newman et al. (43) Pub. Date: Jun. 13, 2013 (54) ENTEROBACTER SP- 638 AND METHODS OF Publication Classification USE THEREOF (51) Int. Cl. (75) Inventors: Lee Newman, Camillus, NY (US); AOIN 63/00 (2006.01) Daniel van der Lelie, Chapel Hill, NC (52) U.S. Cl. (US); Safiyh Taghavi, Chapel Hill, NC CPC ...................................... A0IN 63/00 (2013.01) (US) USPC .......................................................... SO4/117 (73) Assignee: BROOKHAVEN SCIENCE ASSOCATES/BROOKHAVEN NATIONAL LABORATORY, Upton, (57) ABSTRACT NY (US) The present invention relates to a novel species of Entero (21) Appl. No.: 13/634,135 bacter, Enterobacter sp. 638, and to its use in connection, for example, with a method for increasing growth in a plant, (22) PCT Fled: Mar. 10, 2011 increasing biomass in a plant, increasing fruit and/or seed productivity in a plant, increasing disease tolerance and/or (86) PCT NO.: resistance in a plant, and increasing drought tolerance and/or S371 (c)(1), resistance in a plant, as compared to a control or wild-type (2), (4) Date: Jan. 3, 2013 plant grown under identical conditions without application of the inventive method or composition. The methods include Related U.S. Application Data applying an effective amount of a composition, which (60) Provisional application No. 61/313,415, filed on Mar. includes an isolated culture of Enterobacter sp. 638, to the 12, 2010. plant. 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This application claims the benefit of U.S. Provi method includes applying a composition to the plant in an sional Application No. 61/313,415, filed Mar. 12, 2010, amount effective for increasing disease tolerance in the plant. which is incorporated herein by reference in its entirety. The composition includes an isolated culture of Enterobacter 0002 This invention was made with Government support sp. 638. under contract number DE-AC02-98CH10886, awarded by 0014. Other objects advantages and aspects of the present the U.S. Department of Energy. The Government has certain invention will become apparent from the following specifi rights in the invention. cation and the figures. BACKGROUND OF THE INVENTION BRIEF DESCRIPTION OF THE DRAWING(S) 0003. The present invention relates to a novel species of Enterobacter, and to its use in connection with, among other 0015 FIG. 1 is a 16S phylogenetic analysis of the Entero things, plant growth and development. bacter sp. 638 strain. 0004 Changes in the Earth's climate can be expected to 0016 FIG. 2 is a circular representation of the Entero have a strong effect on agricultural productivity. For example, bacter sp. 638 chromosome. Circles displayed (from the out increases in emissions from fossil fuel combustion are con side): the GC percent deviation (GC window-mean GC) in a sidered to have affected the Earth's climate, which have made 1000-bp window, predicted CDSs transcribed in the clock the production of biofuels from renewable resources more wise direction, predicted CDSs transcribed in the counter desirable. Another way in which climate change is expected clockwise direction, CDS in clockwise and counterclockwise to impact agricultural productivity is by increasing tempera direction colored according to their COG classes, the position tures and by affecting rainfall patterns. of all the palindromic repeats, the position of the 100 palin 0005. Although an increased demand of agricultural dromic repeats (CCCTCTCCCXXCX)GGGAGAGGG) resources in the production offeedstocks for biofuel produc (SEQID NO: 1), GC skew (G+C/G-C) in a 1000-bp window, tion is desirable, this increased demand is balanced by a and coordinates in kilo bases pair. Syntenic regions compared simultaneous increased demand for food to feed a still grow with E. coli K12 are shown with genes displayed in orange, ing world population. while genes displayed in purple correspond to non Syntenic 0006. Therefore, there is a need for sustainable practices region. Arrows indicate to putative functions of genes located that can be used to optimize the production of food and in regions that are not in synteny with E. coli K12 (for further biofuel feedstocks. Such practices would optimally increase detail on gene content for each regions see Table 1). A syn overall plant productivity in a Sustainable manner, increase tenic region is defined by a minimum of three consecutive drought tolerance in plants so that crops and feedstocks can genes that are present in a bacterial genome sequence, and withstand major fluctuations in rainfall patterns, and increase that show a similar genetic organization as for the same genes tolerance to pathogen infections in plants. in other bacterial genomes. 0017 FIG. 3 is a circular representation of the Entero SUMMARY OF THE INVENTION bacter sp. 638 plasmid pleNT638-1. Circles displayed from 0007. In one aspect, the invention relates to an isolated the outside: subdivision of pleNT-01 group of function, gene culture of Enterobacter sp. 638. annotation, the GC percent deviation (GC window-mean 0008. In another aspect, the invention relates to an inocu GC) in a 1000-bp window, predicted CDSs (red) transcribed lant for a plant. The inoculant includes an isolated culture of in the clockwise direction, predicted CDSs (blue) transcribed Enterobacter sp. 638 and a biologically acceptable medium. in the counterclockwise direction, GC skew (G+C/G-C) in a 0009. In yet another aspect, the invention relates to a 1000-bp window, transposable elements from IS elements method for increasing growth in a plant. The method includes (pink) and pseudogenes (grey). Toxinfanti T toxin (TA) sys applying a composition to the plant in an amount effective for tems are shown with an asterisk (*). increasing growth in the plant, wherein the composition 0018 FIG. 4 depicts growth indexes for poplar cuttings includes an isolated culture of Enterobacter sp. 638. inoculated with different endophytic bacteria. Growth 0010. In a further aspect, the invention relates to a method indexes were determined 10 weeks after the inoculating and for increasing biomass in a plant. The method includes apply planting of the cuttings in Sandy soil. Per condition, seven ing a composition to the plant in an amount effective for plants were used. Plants were grown in the greenhouse. Non increasing biomass in the plant. The composition includes an inoculated plants were used as references. Bars indicate stan isolated culture of Enterobacter sp. 638. dard errors. Growth indexes were calculated as (Mt-MO)/MO 0011. In yet a further aspect, the invention relates to a after 10 weeks of growth of inoculated and non-inoculated method for increasing fruit and/or seed productivity in a plants. MO, plants weight (g) at week 0; Mt, plants weight plant. The method includes applying a composition to the (g) after 10 weeks. The statistical significance of the plant in an amount effective for increasing fruit and/or seed increased biomass production of inoculated plants, compared productivity in the plant. The composition includes an iso to that of non-inoculated control plants, was confirmed at the lated culture of Enterobacter sp. 638. 5% level (**) using the Dunnett test. 0012. In an additional aspect, the invention relates to a (0019 FIG. 5 shows the effects of Enterobacter sp. 638 on method for increasing disease tolerance in a plant. The the shoot and root formation of poplar DN-34. Plants were method includes applying a composition to the plant in an incubated hydroponically in half-strength Hoagland's solu US 2013/0150240 A1 Jun. 13, 2013 tion in the absence (Control) or presence (638) of strain 638. face-sterilized root and stem samples taken from hybrid pop Root and shoot development are presented after 1 (A) and 10 lar tree H1 1-11 that were grown in a silty loam soil with (B) weeks. groundwater below it that was contaminated with carbon 0020 FIG. 6 shows the total weight of harvested tomatoes tetrachloride or trichloroethylene.