(19) TZZ _¥_T (11) EP 2 791 366 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12R 1/01 (2006.01) A01H 3/00 (2006.01) 26.04.2017 Bulletin 2017/17 (86) International application number: (21) Application number: 12805902.9 PCT/US2012/070036 (22) Date of filing: 17.12.2012 (87) International publication number: WO 2013/090884 (20.06.2013 Gazette 2013/25) (54) BRADYRHIZOBIUM STRAINS BRADYRHIZOBIUM STÄMME SOUCHES DE BRADYRHIZOBIUM (84) Designated Contracting States: • SUGAWARA MASAYUKI ET AL: "Functional Role AL AT BE BG CH CY CZ DE DK EE ES FI FR GB of Bradyrhizobium japonicum Trehalose GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Biosynthesis and Metabolism Genes during PL PT RO RS SE SI SK SM TR Physiological Stress and Nodulation", APPLIED ANDENVIRONMENTAL MICROBIOLOGY, vol. 76, (30) Priority: 16.12.2011 US 201161576470 P no. 4, February 2010 (2010-02), pages 1071-1081, XP002694519, (43) Date of publication of application: • STREETER J G: "Effect of trehalose on survival 22.10.2014 Bulletin 2014/43 of Bradyrhizobium japonicum during desiccation.", JOURNAL OF APPLIED (73) Proprietors: MICROBIOLOGY 2003, vol. 95, no. 3, 2003, pages • Novozymes Bioag A/S 484-491, XP002694520, ISSN: 1364-5072 2880 Bagsvaerd (DK) • CYTRYN EDDIE J ET AL: "Transcriptional and • Novozymes Biologicals, Inc. physiological responses of Bradyrhizobium Salem, VA 24153 (US) japonicum to desiccation-induced stress.", JOURNAL OF BACTERIOLOGY OCT 2007, vol. (72) Inventors: 189, no. 19, October 2007 (2007-10), pages • KANG, Yaowei 6751-6762, XP002694521, ISSN: 0021-9193 Christiansburg, VA 24073 (US) • MARY P ET AL: "Differences among Rhizobium • TRAN, Anh melilotiand Bradyrhizobium japonicum strains in Roanoke, VA 24019 (US) tolerance to desiccation and storage at different • SEMONES, Shawn relative humidities", SOIL BIOLOGY AND Salem, VA 24153 (US) BIOCHEMISTRY,PERGAMON, OXFORD,GB, vol. 26, no. 9, 1 September 1994 (1994-09-01), pages (74) Representative: Rasmussen, Preben et al 1125-1132, XP023671147, ISSN: 0038-0717, DOI: Novozymes A/S 10.1016/0038-0717(94)90134-1 [retrieved on Patents 1994-09-01] Krogshoejvej 36 • ZAHRAN H H: "Rhizobia from wild legumes: 2880 Bagsvaerd (DK) diversity, taxonomy, ecology, nitrogen fixation and biotechnology.", JOURNAL OF (56) References cited: BIOTECHNOLOGY 4 OCT 2001, vol. 91, no. 2-3, 4 WO-A1-00/04778 October 2001 (2001-10-04), pages 143-153, XP002694522, ISSN: 0168-1656 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 791 366 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 791 366 B1 • DEAKER ET AL: "Desiccation tolerance of • MABOOD F ET AL: "Methyl jasmonate, alone or rhizobia when protected by synthetic polymers", in combination with genistein, and SOIL BIOLOGY AND BIOCHEMISTRY, Bradyrhizobium japonicum increases soybean PERGAMON, OXFORD, GB, vol. 39, no. 2, 29 (Glycine max L.) plant dry matter production and November 2006 (2006-11-29), pages 573-580, grain yield under short season conditions", XP005783610, ISSN: 0038-0717, DOI: FIELD CROPS RESEARCH, ELSEVIER, 10.1016/J.SOILBIO.2006.09.005 AMSTERDAM, NL, vol. 95, no. 2-3, 15 February • LEPRINCE O ET AL: "Desiccation tolerance: 2006 (2006-02-15), pages 412-419, XP024938505, From genomics to the field", PLANT SCIENCE, ISSN: 0378-4290, DOI: ELSEVIER IRELAND LTD, IE, vol. 179, no. 6, 1 10.1016/J.FCR.2005.04.013 [retrieved on December 2010 (2010-12-01), pages 554-564, 2006-02-15] XP027449540, ISSN: 0168-9452, DOI: 10.1016/J.PLANTSCI.2010.02.011 [retrieved on 2010-02-24] • SUPANJANI S ET AL: "Nod factor enhances calcium uptake by soybean", PLANT PHYSIOLOGY AND BIOCHEMISTRY, GAUTHIER-VILLARS, PARIS, FR, vol. 44, no. 11-12, 1 November 2006 (2006-11-01), pages 866-872, XP027963384, ISSN: 0981-9428 [retrieved on 2006-11-01] 2 EP 2 791 366 B1 Description REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL 5 [0001] This application contains a reference to a deposit of biological material, which deposit is incorporated herein by reference. For complete information see Table 1. FIELD OF THE INVENTION 10 [0002] The present invention relates to isolated Bradyrhizobium bacterium having enhanced characteristics, including but not limited to, enhanced desiccation resistance. BACKGROUND OF THE INVENTION 15 [0003] In order to maintain healthy growth, plants must extract a variety of elements from the soil in which they grow. These elements include nitrogen and the so-called micro-nutrients (e.g., copper, iron and zinc), but many soils are deficient in such elements or they contain them only in forms which cannot be readily taken up by plants (it is generally believed that essential elements cannot be readily taken up by plants unless they are present in dissolved form in the soil).Nitrogen is an essentialelement for mostplants as it playsa rolein the synthesis of amino acids,proteins, nucleotid es, 20 nucleic acids, chlorophyll, co-enzymes and in the overall growth and health of the plant. To counteract such deficiencies, sources of the deficient elements are commonly applied to soils in order to improve growth rates and yields obtained from crop plants. For example, nitrate and/or ammonium are often added to soil to counteract a lack of available nitrogen. [0004] In the field of crop science, it is well known that many cultivated crops require that the soil provide relatively large amounts of nitrogen to the plant. The notable exceptions to those plants requiring nitrogen via the soil are plants 25 from the legume family. [0005] Specifically, leguminous plants are unique from non-leguminous plants in their ability to fix atmospheric nitrogen into ammonia. The ability to fix atmospheric nitrogen into a useable nitrogen source for the plant obviates the need for the plant to obtain nitrogen from the soil. Nitrogen fixation, however, requires a symbiotic relationship between the leguminous plant and native bacterial within the soil. One of the most extensively studied partners in this symbiotic 30 relationship is bacteria belonging to the genus Bradyrhizobium or Rhizobium. Gresshoff, P. (1999). Identification of Plant Genes Involved in Plant-Microbe Interactions. Stacey, G. & Keen, T. (Ed.), Plant-Microbe Interactions (4th ed.) (Ch. 6). St. Paul: APS Press. [0006] Symbiosis is generally achieved through an exchange of complex bidirectional signaling between the plant and the microbe and the microbe and the plant. Typically, plant factors, such as flavonoids and flavonoid like substances, 35 induce colonization of the bacteria into the root nodule of the leguminous plant. (Gresshoff, 1999). Once the bacteria have colonized the root nodule, the bacteria effect morphological changes in the plant, namely root hair curling and the development of a new root organ - the nodule. (Gresshoff, 1999). The nodule permits the establishment of a new physiological environment for the nodule inducing bacteria to differentiate into a nitrogen-fixing endosymbiont, or bac- teriod, for the colonized plant. (Gresshoff, 1999). 40 [0007] In order to assist with the symbiotic exchange of bi-directional signaling between the plant and microbe, bacteria, such as Bradyrhizobia sp., are often coated on a seed. To prolong the viability of the microbe on the seed, it is desirable that the microbe be tolerant to desiccation and dry environmental conditions generally. [0008] Sugawara Masayuki et al., Applied and Environmental Microbiology, vol. 76, no. 4, Feb 2010, pages 1071-1081 shows trehalose biosynthesis pathways and their effect on dessication tolerance, but do not disclose the specific isolated 45 Bradyrhizobium japonicum strains of the present invention or the specific relative improved desiccation resistance. [0009] Streeter J G, Journal of Applied Microbiology 2003, vol. 95, no. 3, pages 484-491 shows the effect of trehalose on survival of B. japonicum upon dessication, but do not disclose the specific isolated Bradyrhizobium japonicum strains of the present invention or the specific relative improved desiccation resistance. [0010] Cytryn Eddie J et al., Journal of Bacteriology, vol. 189, no. 19, October 2007, pages 751-6762 shows gene 50 expression profiles in B. japonicum in response to dessication-induced stress, but do not disclose the specific isolated Bradyrhizobium japonicum strains of the present invention or the specific relative improved desiccation resistance. [0011] Mary P et al., Soil Biology and Biochemistry, vol. 26, no. 9, pages 1125-1132, shows that these is natural variation in dessication tolerance at different humidities of B. japonicum, but d not disclose the specific isolated Bradyrhizo- bium japonicum strains of the present invention or the specific relative improved desiccation resistance. 55 [0012] There remains a need for microbes with enhanced desiccation resistance. 3 EP 2 791 366 B1 SUMMARY OF THE INVENTION [0013] Described herein are novel bacterial strains having enhanced desiccation resistance, especially when the novel strains are compared to its parental strain, e.g., Bradyrhizobium sp., parental strain USDA 532C. The inventors have 5 isolated and tested a significant number of bacterial strains for their desiccation resistance properties. [0014] As disclosed throughout, the isolated strains are strains of the genus Bradyrhizobium spp. In particular, the isolated strains are strains ofBradyrhizobium japonicum. Even more particularly, the isolated strains are isolated Bradyrhizobium japonicum strains selected from the group consisting of: 10 the strain having the deposit accession number NRRL B-50608; the strain having the deposit accession number NRRL B-50609; the strain having the deposit accession number NRRL B-50610; the strain having the deposit accession number NRRL B-50612, or a combination of at least two or more of the above deposited strains.