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`
`US009205115B2
`
`(12) United States Patent
`Jacobsen
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 9,205,115 B2
`Dec. 8, 2015
`
`(54) BAcILLUS ISOLATES AND METHODS OF
`THEIR USE TO PROTECT AGAINST PLANT
`PATHOGENS AND VIRUS TRANSMISSION
`
`(71) Applicant: MONTANA STATE UNIVERSITY,
`Bozemasa, MT (US)
`
`(72)
`
`(73)
`
`Inventor: Barn’ J. Jacobsen, Bozeman, MT (US)
`
`Assignee: Montana State University, Bozeman,
`MT (US)
`
`(C) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjustcd under 35
`U.S.C. 154(b) by 0 days.
`
`(21)
`
`AppI. No.: 13/966,565
`
`(22)
`
`Filed:
`
`Aug. 14, 2013
`
`(65)
`
`Prior Publication Data
`
`US 2014/0056864Al
`
`Feb. 27, 2014
`
`Related U.S. Application Data
`(63) Continuation of application No. 13/175,614, filed on
`Jul. 1, 2011, now Pat. No. 8,524,222, which is a
`continuation-in-part of application No. 12/557,975,
`filed on Sep. 11,2009, now Pat. No. 8,025,875, which
`continuation-in-part
`application No.
`is
`a
`of
`11/361,283, filed on Feb. 24, 2006, now abandoned.
`
`(51)
`
`Int.Cl.
`A6IK 35/74
`AOIN 63/02
`CI2R 1/07
`(52) U.S. Cl.
`CPC
`
`(2015.01)
`(2006.01)
`(2006.01)
`
`A61K35/74 (2013.01); ,401N63/02
`(2013.01); CI2R 1107 (2013.01)
`(58) Field of Classification Search
`None
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`3.818,104 A
`6,03 1,153 A •
`8.025,875 B2
`8.246,965 B2
`8.524,222 B2
`2005/0244394 Al *
`2006/0029576 At *
`2007/0224 179 Al
`2010/0092442 Al
`2011/0318386 Al
`2012/0003197 Al
`2014/0056864 At *
`
`6/1974 Zielinski
`2/2000 Ryals et al
`9/2011 Jacobsen et al.
`Jacobsen et al.
`8/20 12
`9/2013 Jacobsen et al.
`11/2005 DeChant et al
`2/2006 Huang et nI
`9/2007 Jacobsen et al.
`4/2010 Jacobsen ct al.
`12/2011 Jacobsen et al.
`1/2012 Jacobsen et at.
`2/2014 Jacobsen
`
`OTHER PUBLICATIONS
`
`800/279
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`424/93.4
`
`424/93.46
`
`Barbagus et al. (Biological Control. 2004; 30: 342-35o).
`Neher, Oliver Thomas. Disease control and plant defense pathways
`inducedby Bacillus mojavcnaia isolate 203-7 andBacillos mycoides
`isolate BmJ. Diss. Montana State University-Bozeman, College of
`Agriculture, 2008*
`
`Kloepper et al. (Phytopathology. 2004; 94(11): l259l266).*
`Alslrdm, S., “Induction of Disease Resistance in Common Bean
`Susceptible to Halo Blight Bacterial Pathogen After Seed Bacterisa
`lion With Rbizosphere Pseudomonads’ Journal of Genetic and
`Applied Microbiology, 37:495-501 (1991), USA.
`Alvarez et al., “Reactive Oxygen tntermediates Mediate a Syatesuic
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`92:773-784, Mar. 20, 1998.
`Andrews, J.H., “Biological Control in the Phyllosphere,” Annual
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`Bargabus. RU, et at.. “Bacillus Mycoides Isolate Bac J Elicits an
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`2003, Charlotte, NC, tISA.
`Bargabus. R.L., et al., “Characterisation of Systemic Resistance in
`Sugar Beet Elicited by a Non-Pathogenic, Phyllospehere-Colonizing
`Bacillus Mycoides, Biological Control Agent:’ (2003) Physiological
`and Molecular Plant Patholgy (2002) 61, 289-298, USA.
`Bargabus, R.L.. et al,, “Elicitation of ISR by a Nonpathogenic Phyl
`losphere Inhabiting Bacterium,” APS Annual Meeting, Aug. 25-29,
`2001 Postet Abstract, Charlotte, NC, USA.
`Bargnbus. R.L.,etal,, “Host-response Based ScreeningofBiological
`Control Agents:’ APS Abstracts of Presentations (2002) APS Annual
`Meeting, Jul. 27-31, 2002, Midwest Express Center, Milwaukee,
`Wisconsin, USA.
`Bargabus. R.L.. et al., “Oxidative Burst Elicited by Bacillus
`Mycoidea Isolate Bac J, a Biological Control Agent, Occurs inde
`pendently of Hypersensitive Cell Death in Sugar Beet,” American
`Phytopathological Society vol. 16, No. 12, 2003, pp. 1145-1153,
`USA.
`Bargabus. R.L., et al., “Screening for the tdcntification of Potential
`Biological Control Agents That Induce Systemic Acquired Resis
`tnncc in Sugar Beet,” Department of Plant Sciences and Plant Pathol
`ogy. Montana State University, Biological Control, 30:342-350
`(2004), USA.
`Bargabus-Larson, R.L., et al,, “Biocontrol Elicited Systemic Resis
`tance in Sugarbeet is Salicylic Acid Independent and NPRI Depen
`dent:’ USDA, .A.griculniral Research Service, Sugarbeet Research
`Unit, 1701 Centre Avenue, Fort Collins, Colorado and Montana State
`tJniveraity, Biocnntrol Elicited Systemic Reaistance,pp. 17-33, Jan.-
`Jun. 2007, USA.
`Chen et al,, “Biological control ofgrapevinecrown gall: purification
`and partial characterisation of an antibacterial substance producedby
`Rabncllaaguatilis strain HX2”, Eur. J. Plant Phathol. 2009. 124 :427-
`437.
`
`(Continued)
`
`Primary Examiner
`Shanon A Foley
`(74) Attorney, Agent, or Fin,, — Cooley LLP
`
`(57)
`
`ABSTRACT
`
`Methods of inducing systemic acquired resistance to infec
`tion in a plant are provided. The methods comprise applying
`a composition compr1sing a Bacillus contrul agent to said
`plant wherein said plant
`is capable of producing defense
`proteins. Also provided are, methods for controlling one or
`more plant diseases, methods for preventing plant virus trans
`mission, methods for preventing and/or treating soil-home
`plant pathogens using the Bacillus control agent of the
`present
`invention, and methods of generaling bacterial
`spores. In addition, synergistic biocontrol combinations and
`methods of using the sasne are provided.
`
`16 Claims, 6 DrawIng Sheets
`
`o ma,
`
`>
`
`Ez -
`
`._d
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`
`p.
`
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`C.-
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`SYNGENTA EXHIBIT 1040
`Syngenta v. UPL, PGR2023-00017
`
`

`

`Us 9,205,115112
`Page 2
`
`(56)
`
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`‘Effective Control of Ariuywonn, Spodoptera exigila
`Cheng ci aI.,
`(Hubner). on Green Onion by the OvicidaL Action of Bifenthrin”.
`Jour. Agric. Rca. China, 1988, 37(3) 320-327.
`Doke, N., “Generation of Superoxide Anion by Potato Tuber
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`Components of Phytophthora Infcsrans and Specific inhibition ofthe
`Reaction by Suppressors of Hypersensitivity,” Physiological Plant
`Patholom. 23:359-367 (1983). USA.
`Enya et al.. “Culturable Leaf-Associated Bacteria on Tomato Plants
`and Their Potential as Biological Control Agents”, Microbial Ecol
`ogy, 2007. 53:524-536.
`Gasbe]olto et at, ‘Efikacy of phosphonic acid, metaiaxyl-M and
`copper hydroxide aainsl Phytophthora rajnonJm in vitro and in
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`plants”. Plant Pathology, 2008
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`Gust es al.. “Biosechnologicat concepts for ioptoving plant innate
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`the Third International Symposium on Biological Control of
`Arthropoda, 2008,403-415.
`Jacobsen etal., “Cercospora Leaf Spot Control Research at Sidney,
`M’f IN 1998”, Jan. 2000, 1999 Sugarbeet Research and Extension
`Reports, vol. 30, pp. 287-288.
`Jacobsen et at., “Fungicide and Biological Control Alternatives to
`TPTH forCercosporaLeafSpotControl”, Jan. 1999. 1998 Sugarbeet
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`Jacobsen et al. “Integrated Managemeni ofCercospora Leaf Spot”,
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`Jaccbsenet al.. “IntegrariooofBacillos sp. biological seedtreartnents
`with apron’thiram and apron-thiramtachigaren seed treabnents for
`biological control of pythium and aphanomyces seedling diseases”,
`and “tntegrated Management strategies for Rhizoctonia Crown and
`Root Rot”, Jan. 2002, 2001 Sugarbt Research and Extension
`Reports, vol. 32, pp. 262-265.
`Jacobson et al., “Management of Cercosposa Leaf Spot in Wcrterss
`North Dakota and Eastern Montana”, Jan. 2001. 2000 Sugasbeet
`Research and Extension Reports, vol.31. pp.273-276.
`Jacobsen. B.J.. et al., “Commericalization ofl3acillus Mycoides Iso
`late BnsJ as a Broad Specirum Biological Plant Disease Control
`Agent’ Phytopathology 97:S50, USA.
`Jacobsen, B.J., ctal., “The Roleofflacillus-bascd Biological Control
`Agents in Integrated Pest Management Systems,” Abstracts of Spe
`cial Session Presentations APS Annual Meeting Aug. 9-13, 2003,
`Charlotte, NC, USA.
`Johnson, C., etal., ‘SalicylicAcidandNPRl Lttducethe Reqruitment
`of Trans-Activating TGA Factors to a Defense Gene Promoter in
`Arabidopsis,” The Plant Cell, vol. 15, 1846-1858 (2003), USA.
`Kloepper et al., “Induced Systemic Resistance and Promotion of
`Plant Growth by Boci!Ius spp.”, 2004, Phytopathology. 94:1259-
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`860 (1982), USA.
`Larson, B.J., et al. “Integrating Fungicides and a Bacillus Mycoides
`Biological Control Agent to Manage Cercospora Leaf Spot Resis
`tance to Fungicides:’ APS Abstracts of Presentarions, APS 2002
`Annual Meeting, Jttl. 27-31,2002, Midwest ExpressCenter, Milwau
`kee, Wisconsin, USA.
`
`Matsuda es al. “Control of the Bacterial wilt of Tomato Plants by a
`Derivative of 3-Indolepropionic Acid Based on Selective Actions on
`Ralstonia solanaceanun”, Journal of Agriculteral Food Chemistry,
`1998, 46:4416.4419.
`Neber et al., ‘Defense pathways activated by Bacillus mojavensis
`isolate BmJ as elucidated by
`isolate 203-7 and B. mycoides
`Arabidopsis mutants”, Abstract of Presentations, Sep. II, 2008, X”
`meeting of the working group, biological control of firtsgal and bac
`terial plant pathogens, International Organization for Biological and
`Integrated Control ofNoxious Animals and Plants. Interla]cen. Swit
`zerland.
`Neher. O.T.,et al., “The Control ofAnthracnoseofCucurbits Caulsed
`by GIoo,eretlo cbrguloro Var. Orbiculare by Foliar Applications of
`Bacillus Mycoides Isolate BmJ,” Phytopathology 97: 583. USA.
`Pieterse, C.M.J.,etai.. “Sysrcusic Resistance inArabidopsislnducnl.
`by Rioconsrol Bacieria is Independent of Salicylic Acid Accumula
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`8:t2151237tt996). USA.
`Sampson, MN., et al., “Involvetisent of Cbitinases of Bacillus
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`Santos, et al. “In aCompalible Plant-pathogen Interaction, a Single,
`Rapid Burst of Hydrogen Peroxide is Observed,” MPMI vol. 14, No.
`1, 2001, pp.86-89. Publication No. M-2000-l 117-01W, TheAsneri
`can Phytopathological Society (2001), USA.
`Sequeira, L.. “Mechanisms of Induced Resistance in Plants:’ Annual
`Review of Microbiology, 37:5, 1-79 (1983), USA.
`Silva el al,. ‘Induction of Systemic Resistance by Bacillus cereus
`Againsi Toni.alo Foliar Diseases Under Field Conditions,” J.
`Phytopathology 152:371.375 (2004).
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`Resistance to Pathogens’ Induced Plans DefensesAgainst Pathogens
`and Herbivores (:\.A. Agrawal, S. tuzun, and P. Bent, ecIt.) SI. Patti:
`APS Picas, 357-369 (1999), USA.
`Wei, G., et al.. “Induction of Systemic Resistance of Cucusnber to
`Collctotrichusn Orbiculare by Select Strains of Plant Growth-pro
`moting Risizobacteria.” Phytopathology, 81:1508.1512 (1991),
`USA.
`Zaitlin et al., “Advances in Understanding Plant Vinises and Virus
`Diseases”, Anmtal Review in Phytopathology, 2000,38:117-143.
`Zietlow, 0 T., nt al.. “Induced Systemic Resistance in Cucumber to
`Glooterella cingsslala Var. Otticulare and Pseudomans Syringae pv.
`Isolate BacJ and Bacillus
`Lachrymasis by Bacillus Mycoides.
`Pusniitts, Isolate MSU 203-7.”APS Abstracts of Presentations, 2004
`Annual Meeting of the A_’nerican Phytological Society. USA.
`Jacobsen et al., “Integrated Control of Soilborne Diseases on Sugar
`Beet with Antagonistic Bacteria and Fungscidcs”, 1997 Sugarbeet
`Research and Extension Reports, vol. 28, (1997).
`Jacobsen, B.J., et al., “Commercialization of Bacillus Mycoides iso
`late BnU as a Broad Spectrum Biological Plant Disease Control
`Agent,” Phytopathology 97:S50, USA (2003).
`Jacobsen ct al., “Seed Treatments for Control of Pythium and
`Apbanomyces Black Root Rot”, 1998 Sugasbeet Research and
`Extcnsion Reports, vol. 29, (1998).
`Kloepper et nI., “Induced Systemic Resistance and Promotion of
`Plant Growth by BocilIus spp.”, Phytopathotogy. 94:1259-1266
`(2004).
`Neher,O.l., etai,. “The Control ofAntlsracnose ofCncurbitsCaulsed
`by Glvgme,rIlo chtgulato Var. Orbiculare by Foliar Applications of
`Bacillus Mycoides Isolate BmJ:’ Phylopathology 97: S83, USA
`(2007).
`
`* ciled by examiner
`
`

`

`U.S. Patent
`
`Dec. 8,2015
`
`Sheet 1 of 6
`
`US 9,205,115 B2
`
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`
`

`

`U.S. Patent
`
`Dec. 8,2015
`
`Sheet 2 of 6
`
`US 9,205,115 B2
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`
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`
`

`

`U..S. Patent
`
`Dec. 8,2015
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`US 9,205,115 B2
`
`I
`BACILLUS ISOLATES AND THODS OF
`THEIR USE TO PROTECT AGAINS’f PLANT
`PATHOGENS AND VIRUS TRANSMISSION
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application claims priority to U.S. patent application
`Sen No. 13/175,614, filed Jul. 1, 2011, now U.S. Pat. No.
`8,524,222, which is itself a continuation-in-part of U.S.
`patent application Ser. No. 12/557,975 filed on Sep. 11,2009,
`00w U.S. Pat. No. 8.025,875, which is a continuation-in-part
`of U.S. patent application 5cr. No. 11/361,283 filed on Feb.
`24, 2006, each of which is hereby incorporated by reference
`in its entirety for all purposes.
`
`GOVERNMENT RIGHTS STATEMENT
`
`This invention was made with government support under
`grant number 2001-35316-11109 awarded by United States
`Department of Agriculture (IJSDA)/CSREES, and under
`grant number 2005-33610-16085 awarded by USDA. The
`govermnent has certain rights in the invention.
`
`FIELD OF THE INVENTION
`
`This invention generalty relates to methods of inducing
`pathogen rcsistance in plants, such as inducing systemic
`acquired resistance to infection in plants. In one aspect, this
`invention relates to methods of inducing systemic acquired
`resistance to infection in plants comprising applying a Bacil
`lus control agent comprising Bacillus Inojavensis isolate
`203-7 and/or Bacillus ,,roides isolate BmJ to one or more
`plants. The present invention also relates to the field of bac
`terial spore production for hiopesticides. In one aspect, the
`invention relates to novel methods of generating
`present
`Bacillus spores.
`
`BACKGROUND OF THE INVENTION
`
`Effective biological control of plant diseases with epi
`phytic microbes has been documented for numerous phyllo
`sphere- and rhizosphere-inhabiting organisms. Foliar bio
`logical control agents include yeast and filamentous fungi
`(see Hofstein R and A. Chapple, ‘Commercial development
`ofbiofungicides,” Biopesticides: Use and Delivery (Hall F It,
`MennJ J, eds.), Totowa: Humana Press (1999); and Sutton, 3.
`C. and G. Peng, “Manipulation and vectoring of biocontrol
`organisms to manage foliage and fruit diseases in cropping
`systems,” Annual Review of Phytopathology. 31:473-493
`(1993)) as well as bacteria; including both gram (—) species
`such as Erwinia sp. and Pseudomouas sp. (seeAndrews, I. H.,
`“Biological control in the phyllosphere,” Annual Review of
`Phytopathology, 30:603-635 (1992)), and gram (+) organ
`isms such as Bacillus sp. Sec Kokalis-Burelle, N., P. A. Back
`man, R Rodriquez-Kabana. and L. D. Ploper, ‘Potential for
`biological control of early leafspot of peanut using Bacillus
`cereus and chitin as foliar mnendments’ Biological Control,
`2:321-328 (1992). Biological control agents applied to the
`rhizosphere include Pseudomonads (see Alstroin, S., “Induc
`tion of disease resistance in common bean susceptible to halo
`blight bacterial pathogen after seed hacterisation with rhina
`sphere pseudomonads,” Journal of Genetic and Applied
`Microbiology, 37:495-501
`(1991): van Peer, R, G.
`J.
`Niemann,andB. Schippers, “Inducedresistanceandphytoal
`cxin accumulation in biological control of fusariuni wilt of
`carnation by Pseudoo,onasa sp. strain WCS417r,” Phytopa
`
`2
`thology. 81:728-734(1991); andvanLoonL. C. andC. M. I.
`Pieterse, “Biological control agents in signaling resistance,”
`Biological Control of Crop Diseases (Gnanamanickan S S,
`ed.), New York: Mercel Dekker, Inc, 486 (2002)) as well as
`5 Bacillus sp. (see Zhang, S., M. S. Reddy, N. Kokalis-Burelle,
`L. W. Wells, S. P. Nightengale, and J. W. Klneppesi “Lack of
`induced systemic resistance in peanut to late leaf spot disease
`by plant growth-promoting rhizobacteria and chemical elici
`tors,” PlaotDisease, 85(8):879-884 (2001); andMurphy, 3. F.,
`to G. W. Zehnder, D. J. Schuster, E. J. Sikora, J. E. Polstoa, and
`J. W. Klnepper, “Plant growth-promoting rhizobacterial
`mediated protection in tomato against Tomato mottle virus,”
`Plant Disease, 84(7):779-784 (2000)) that are classically
`referred to as plant growth-promoting rhizobaeteria. For the
`is most part biological disease control
`is attrihuted to direct
`antagonism against the pathogen via production ofantibiotics
`or hydrolytic enzymes, or through competition for nutrients.
`See Weller. D. M., “Biological control of soil-borne plant
`pathogens in the rhizosphere with bacteria?’ Annual Review
`20 of Phytopathology. 26:379-407 (1988). However, plant
`growth-promoting rhizobacteria and rhizosphere inhabiting
`fimgi have been shown to stimulate the induction of systemic
`resistance responses within the plant. See van Peer. R. G. J.
`Niemann. and B. Schippers, “Induced resistanceandphytoal
`25 exin accumulation in biological control offi.sat-iun; wilt of
`carnation by Pseudon,onasa sp. strain WCS417r?’ Phytopa
`thology, 81:728-734 (1991); Wei. G. 3. W. Kloepper. and S.
`Tuzun, “Induction of systemic resistance of cucumber to
`Colleioirichum orbiculare by select strains of plant growth-
`30 promoting rhizobacteria,” Phytopathology, 81:1508-1512
`(1990; van Loon, L. C. and C. M. J. Pieterse, “Biological
`control agents in signaling resistance?’ Biological Control of
`Crop Diseases (Gnanamanickan. S.S., ed). New York: Mer
`eel Dekker,
`Inc. 486 (2002). All publications mentioned
`35 abnve are incorporated herein by reference in their entireties
`for all purposes.
`Systemic induced resistance (SIR) has been described in
`many plant systems, most notably tobacco, bean, tomato,
`cucumber, and Arabidopsis thaliana. See Ross, A F., “Local-
`ized acquired resistance to plant virus infection in hypersen
`sitive hosts,”Virology, 14:329-339(1961); Kuc, J., “Induced
`immunity to plant disease;’ BioScience, 32:854-860(1982);
`Ryals, J.A.,U. El. Neuenschwander, M. G. Willits,A, Molina,
`H. Y. Steiner, and M. D. Hunt, “Systemic acquired resis
`tance,”The PlanECell. 8:1809-1819(1996); andvanLoon, L.
`C. and C. M. 3. l’ietetse, “Biological control agents in signal
`ing resistance.” Biological Control of Crop Diseases (Gnana
`maniekan. S. S., ed). New York: Mercel Dekker, Inc. 486
`(2002). The broad-spectrum resistance makes an otherwise
`So susceptible plant resistant
`to a wide array of subsequent
`pathogen attacks. See Kuc, J. “Induced immunity to plant
`disease;’ BioScience. 32:854-860(1982); and Hutcheson, S.
`W., “Current concepts of ‘active defense in plants.” Annual
`Review of Pbytopathology, 36:59-90 (1998). Elicitation of
`systemic disease resistance in plants has thus far been
`achieved through treatment by three types of stimuli: necro
`tizing pathogens (see Pieterse, C. M. J., S.C.M. van Wees. E.
`Hoffiand, J. A. van Pelt, and L. C. van Loon, “Systemic
`resistance in Arabidopsis induced by biocontrol bacteria is
`60 independent ofsalieylic acid accumulation andpathogenesis
`related gene expression;’ The Plant Cell, 8:1225-1237
`(1996); Ross, A F., “Localized acquired resistance to plant
`virus infectiuninhypersensitivehusts,”Vbulogy, 14:329-339
`(1961); Ross. A F., “Systemic acquired resistance induced by
`localized virus infection in plants;’ Mrology. 14:340-358
`(1960; and Kuc, J., “Induced innnunity to plant disease?’
`BioScience. 32:854-860(1982)), secondary signal molecules
`
`40
`
`45
`
`55
`
`65
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`

`

`US 9,205,115 B2
`
`15
`
`20
`
`3
`(Le. salicylic acid, SA) (see White, R. F., “Acelylsalicylic
`acid (aspirin) induces resistance to tobacco mosaic virus in
`tobacco;’ \‘iro]ogy. 99:410-412 (1979)) and their functional
`INA (see
`acid,
`(e.g. 2,6-dichloroisonicotinic
`analogs
`Meiraux, j, p., a Alil-Goy, T. Staub, J. Speich,A Sieineinann.
`3. Ryals, and H. Ward, “Induced resistance in cucumber in
`response to 2,6-dichloroisonicotinic acid and pathogens.”
`Advances in Molecular Genetics of Plain-Microbe Interac
`tions. Vol. I. (1-i. Hennecke. D. R S. Venna, eds.), Dordrecht:
`Kiuwer Academic Publishers, 432-439 (199 1)) and aciben
`zotar-S-methyl. ASM (see Tally, A, M. Oostendorp, K. Law
`ton, T. Staub. and B. Bassi, “Commercial development of
`eticiters of induced resistance to pathogens,” Induced Plant
`Defenses Against Pathogens and Herbivores (A\ Agrawal, S.
`Tuzun, and E. Bent, eds.) St. Paul: APS Press, 299-318
`(1999)), and plant growth-promoting rhizobacteria introdttc
`lion into the rhizosphere. See Alstrom, S., “Induction of dis
`ease resistance in common bean susceptible to halo blight
`bacteria] pathogen after seed bacterisation with rhizosphere
`pseudomonads’ Journal of Genetic and Applied Microbiul
`ogv,37:495-501 (1991); vant.oon. L. C. and C. M. I. Pieterse.
`‘Biological control agents in signaling resistance.” Biologi
`cal Control of Crop Diseases (Gnnnamanickan, S. S., ed),
`New York; Meted Dekker. Inc. 486 (2002); Wei, 0., 3. W.
`Kloepper, and S. Tnzun, “Induction of systemic resistanceof 25
`cucumber to C’olletob-ichu,n orbiculare by select strains of
`plant growth-promoting rhizobactcria,” Phytopathology.
`81:1508-1512 (1991); Zhang, S., M. S. Reddy, N. Kokalis
`Borelle, L. W. Wells. S. P. Nightcogale, and J. W. Kloepper,
`“Lack of induced systemic resistance in peanut to late leaf
`spot disease by plant growth-promoting rhizubacieria and
`chemical elicitors,”PlantDisease, 85(8):879-884 (2001); and
`Murphy, J. R, G. W. Zehnder, D. I. Schuster, H. I. Sikora, I.E.
`Poiston, and J. W. Kloepper, “Plant growth-promoting rhizo
`bacterial mediated protection in tomato against Tomato
`mottle virus,” Plant Disease, 84(7):779-784 (2000). Addi
`tionally, oomycete and fungal hyphal wall fragments (see
`Doke, N., “Generation of superoxide anion by potato tuber
`protoplasts during the hypersensitive response to hypbal wall
`components ofPhytophthora infrstans and specific inhibition
`of the reaction by suppressors of hypersensitivity’ Physi
`ological Plant Pathology, 23:359-367 (1983); andAnderson,
`A. J., “Studies on the structure and elicitor activity of flingal
`glucans.” Canadian Journal of Botany, 58:2343-2348
`(1980)). bacterial cell wall fractions (lipopolysaccharidm)
`(see Snueira, L., “Mechanisms of induced resistance in
`planis,” Annual Review of Microbiology, 37:51-79 (1983).
`and phylohorinones (see Cohen. Y., M. Reuveni, and A.
`Baider, “Local and systemic activity of BABA (DL-3-ami-
`nobutyric acid), against Plasniopara viticala in g,rapevmes,”
`European Journal of Plant Pathology, 105(4):35l-36l
`(1999); Oh. Y., Y. Cohen. andY Spiegel, “Local and sys
`temic induced resistance to the root-knot nematode in tomato
`by DL-beta-amittto-n-butyric acid.” Phytopathology, 89(12):
`1138-1143 (1999); and Cohen. Y R., ‘Aminobutyric acid
`Induced Resistance .kgaiost Plant Pathogens’ Plant Disease,
`86(5):448-457 (2002)) have SIR-displayed induction capa
`bility. All publications mentioned above are incorporated
`herein by reference in their entireties fur all purposes.
`Two systemic resistance pnthwas have been described: I)
`systemic acquired resistance, which utilizes salicylic acid as
`a secondary signal molecule and leads to the production of
`pathogenesis-related (PR) proteins (see Delaney. T. P.,
`“Genetic Dissection of Acquired Resistance to Disease,”
`I 3:5-12(1997)) and 2) induced systemic
`Plant, Physiology. I
`resistance, which utilizcsjasmonates and ethyleneas second
`ary signal molecules and controls disease independently of
`
`4
`PR-protein production (see Pieterse, C. M. J., S. C. M. van
`Wces, J. A. van Pelt, M. Knoester, R. Laan, H. Genus, P. 1.
`Weisbeek, and L. C. van Loon, “A Novel Signaling Pathway
`Controlling Induced Systemic Resistance in Arabidopsis’
`S The Plant Cell, 10:1571-1580 (1998)), All publications men
`tioned above are incorporated herein by reference in their
`entireties for all purposes.
`Systemic resistance results in the activation of defenses in
`oninfected parts of the Nan!. As a result, the entire plant is
`to more resistant to infection. The systemic resistance is long
`lasting and often confers broad-based resistance to different
`pathogens.
`One of the issues surrounding systemic resistance is the
`occurrence of necrotic cell death at the site of application of
`the agent that induces systemic resistance.
`Increased societal concerns related to the use ofagrichenii
`cals and genetically modified organisms as a means of man
`aging crop diseases has prompted interest in methods of bio
`logical control. A biological control agent capable of
`inducing systemic resistance would provide a method of
`increasing disease resistance in a plant without the use of
`agrichemicals. Of particular interest would be a biological
`control agent capableof inducing systemicresistance without
`nducing necrotic cell death,
`Thos. a need exists for new biological control agents
`capable of inducing systemic induced resistance In plants. A
`need also exists for new methods of idennifying new biologi
`cal control agents capable of inducing systemic resistance in
`plants.
`Bocillussporescanpotentiallybeusedas biocontrol agents
`for suppressing various plant diseases. See, e.g., Emmert EA
`(1999) Biocontrol of plant disease-a
`B, Handeisman J
`(Gram-) positive perspective. FEMS Microbiol. Lett. 171:1 -
`9; Shoda M (2000) Bacterial control of plant diseases. J.
`35 Biosci. Biocng. 89:515-521; Montesinos H (2003) Develop
`inent, registration and commercialization of microbial pesti
`cides for plant protection. Int. Microbiol. 6:245-252. Spores
`are the preferred form for commercial delivery as spores are
`more efficient and less expensive to produce and more stable
`40 than freeze dried cells. Such biocontrol agents are desirable
`over chen3ical agents, which arc often harmful to Ihc environ
`ment and to humans. However, the current high costs of spore
`production caused by inefficiencies in culturing and fennen
`tation methods have prevented the widespread use ofBacillus
`spores to control plant disease.
`Many attempts have been made to enhance spore yields,
`particularly with Bacillus subtilis cells. See, e.g.. Monteiro S
`(2005) A Procedure for Fligb-Yield Spore Production by
`Bacillus subtilis. Biotechnol. Ping. 21:1026-1031; Hageniati
`so J H, et al.. (1984) Single. chemically defined sporulation
`medium for Bacillus subrilis grow Eli, sporulatioo, and extra-
`J. Bactcriol. 160:438-441;
`cellular protease production.
`Dingman, D Wand Stably, D P (1983) Medium Promoting
`Sponilation of Bacillus larvae and Metabolism of Medium
`55 Components. AppI. Environ. Microbiol. 46(4):860-869: War
`riner, K, and Waites, W. M. (1999) Enhanced Sponitation in
`Bacillus subtilis Grown on Medium Containing Glucose:
`Ribose. Letters in Applied Microbiology 29:97-102: Chen,
`Z., et al., (2010) Greater Enhancement of Bacillus subrilis
`60 Spore Yields in Submerged Cultures by Optimization of
`Medium Composition Through Statistical Experimental
`85:1353-1360.
`Designs. AppI. Microbiol. Biotechnol.
`Researchers have also adapted known spore culture methods
`in attempts to produce spores of Bacillus on’coides. See, for
`ss example. Bowen et al. (Jul. 20, 2002) The Mcasuretnettt of
`Bacillus mycoides Spore Adhesion Using .ktomic Force
`Microscopy, Simple Counting Methods, and a Spinning Disk
`
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`Us 9,205,115 B2
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`5
`Technique. Biotechnology and Binengineering, Vol. 79(2):
`170-179. However, improved inetltods for spore production
`are needed, particularly for other species within the Bacillus
`genus.
`
`SUMMARY OF THE INVENTION
`
`In accordance with the objects outlined above, the preseot
`io
`invention provides methods and compositions useflul
`inducingdisease resistance to infection inn plant, comprising
`applying a Bacillus control agent comprising Bacillus mojav
`isolate ‘203-7’ having accession number NRRL
`crisis
`8-30893 and/or Bacillus rni’coides isolate ‘BmJ’ haviog
`accession number NRRL 8-3089010 the plant. wherein the
`In olle
`is capable of producing defense proteins.
`plant
`embodiment, the disease resistance to infection in the plant is
`systemic acquired resistance. ‘BmJ’ is also known as ‘Doe .1.’,
`Bacillus inycoides isolate 1,11. mj’co ides J, Bacillus uuycoides
`the systemic
`In another embodiment,
`3, or ‘isolate 3”.
`acquired resistance is induced in the plant through a salicylic
`acid independent and jasnionic acid dependent pathway. In
`the systemic acquired resistance is
`another embodiment,
`induced by Bacillus tuycoides isolate Dm1 having accession
`number NRRL 8-30890 in the plant through anNPR1 depen
`dent pathway. In another embodiment, the systemic acquired
`resistance is induced by Bacillus majavensis isolate 203-7
`having accession number NRRL 8-30893 in the plant
`through an NON-EXPRESSOR OP PATHOGENESIS-RE
`LATED GENESI (NPRI) independent pathway. In another
`embodiment, the plant is a monocot, for example, the plant is
`selected from the group consisting ofwheat,Arabodpsis, corn
`In another embodi
`triticale and lily.
`(maize). rice, barley.
`unetit. the plant is a dicot, for example. the plant is selected
`from the group consisting of banana, cucurbit. pecan. soy
`tomato, cucumber. watennelon.
`bean. sunflower, alfalfa,
`potato, pepper, bean, cli.rysattthemtun, and geranium. In
`another embodiment, the infection is caused by any kind of
`infectious (i.e., biottc) agents that affect plants. Examples of
`such agents/pathogens include but are not limited to an agent
`or pathogen selected from the group consisting of bacteria,
`fungi, and vinuses. Examples of specific pathegens to he
`treated using the compositions and methods of the present
`invention include but are not limited to pathogens selected
`from the group consisting of MycaspkaerellaJijiensis, CIa
`dosporiuin carj’igen an,, Glanierella cingulata, Cercospara
`beticola, Pseudornanassvriuge, Erwinia caratovora, Dairy/is
`cinerea, and Fusariu,n salani f. sp. cucurbitae, Alteruaria
`solani, Sclerolinia sclerotic porn, Aliernaria solani, Sclera
`tin Ia sclerotiorum, Xantha,nonas camp es/ris, Pythium aph
`In some other
`anidenuatun,, and Padosphora xaolhii.
`entbudiments, thedisease is associated with plant viruses, for
`example, Potato Vints Y, cucumber mosaic virus, tobacco
`mosaic virus, and squash vein yellowing virus.
`The present invention also provides methods of inducing a
`first systemic acqutred resistance in a plantcomprising apply
`ing a Bacillus contrul agent comprising Bacillus nrojavensis
`isolate 203-7 and/or Bacillus ,,0’coide.c isolate Rns.l to the
`plant, wherein the methods further comprise applying a sec
`uod biological or chemical control agent, and wherein the first
`systemic acquired resntance is induced inthe plantthrough a
`salicylic acid independent andjasmonic acid dependent path
`the first systemic acquired resis
`way. In one embodiment,
`tance is induced by Bacillus mycoides isolate BmJ having
`accession number NRRL 8-30890 in the plant through an
`NPR1 dependent pathway. In another embodiment, the first
`systemic acquired rcsistance is induced by Bacillus nrojav
`crisis isolate 203-7 having accession number NRRL 8-30893
`
`20
`
`25
`
`6
`independent pathway. In
`through an NPRI
`in the plant
`another embodiment, the second biological or chemical con
`trol agent is selected from the group consisting of antifiungal
`agents, antibacterial agents, antiviral agents, and plant acti
`5 vatingeompounds. The second biological orcltemical control
`agent mayor may not also induce the first systemic acquired
`resistattce in the plant and/or induce a second systemic
`acquired resistance in the plant.
`The invention is also directed to ntethods of screening for
`control agents useful
`inducing systemic
`in
`11) biological
`acquired resistance to infection in a plant.
`The present invent