EP0941350A1 - SOUCHES DE $i(PSEUDOMONAS) GENETIQUEMENT MODIFIEES AYANT UNE ACTIVITE DE LUTTE BIOLOGIQUE AMELIOREE - Google Patents

SOUCHES DE $i(PSEUDOMONAS) GENETIQUEMENT MODIFIEES AYANT UNE ACTIVITE DE LUTTE BIOLOGIQUE AMELIOREE

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Publication number
EP0941350A1
EP0941350A1 EP97954359A EP97954359A EP0941350A1 EP 0941350 A1 EP0941350 A1 EP 0941350A1 EP 97954359 A EP97954359 A EP 97954359A EP 97954359 A EP97954359 A EP 97954359A EP 0941350 A1 EP0941350 A1 EP 0941350A1
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EP
European Patent Office
Prior art keywords
strain
biocontrol
gaca
gene
plant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP97954359A
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German (de)
English (en)
Inventor
James Madison Ligon
Dwight Steven Hill
Stephen Ting Lam
Thomas Deane Gaffney
Nancy Reeves Torkewitz
Jill Michelle Stafford
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AGRASOL Inc
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Novartis AG
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Priority claimed from US08/761,258 external-priority patent/US5756087A/en
Application filed by Novartis AG filed Critical Novartis AG
Publication of EP0941350A1 publication Critical patent/EP0941350A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/27—Pseudomonas

Definitions

  • the present invention relates to genetically modified strains of Pseudomonas that have improved biocontrol properties. More specifically it relates to strains that are effective against plant pathogenic fungi.
  • the most efficient strains of biological disease controlling bacteria are fluorescent pseudomonads ( WeBereta/. (1983) Phytopathology, 73: 463469). These bacteria have also been shown to promote plant growth in the absence of a specific fungal pathogen by the suppression of detrimental rhizosphere microflora present in most soils ( Woepperet a/.(1981) Phytopatholog 71: 1020-1024). Important plant pathogens that have been effectively controlled by seed inoculation with these bacteria include Gaemannomyces graminis, the causative agent of take-all in wheat ( Cook era/.(1976) Sol Bbl.
  • Rhizoctonia is a particularly problematic plant pathogen for several reasons. First, it is capable of infecting a wide range of crop plants, and second, there are no commercially available chemical fungicides that are effective in controlling the fungus.
  • pyrrolnitrin A particularly effective antibiotic against fungal p athogens is pyrrolnitrin, which is biosynthesized from tryptophan (Cnangefa/. J.Anlbiot 34: 555-566). Pyrrolnitrin is a phenylpyrrole derivative with strong antibiotic activity that has been shown to inhibit a broad range of fungi (Hommaera/.,So_ Bid. Biochem.21: 723-728(1989); r ⁇ dae*a/.,J.Ar ⁇ tt ⁇ ,ser. 18: 211-219(1965)). Pyrrolnitrin was originally isolated from Pseudomonas pyrrocinia (Arimaetal, J.
  • biocontrol organism An important factor in biological control is the ability of a biocontrol organism to compete in a given environment (Baker etal. (1982) Biological (Itoritid of PlartPatix ⁇ Phytopathobgcal Society, St Paul, Mnn., pages 61-106).
  • Biological Itoritid of PlartPatix ⁇ Phytopathobgcal Society, St Paul, Mnn., pages 61-106.
  • the present invention is drawn to genetically engineered biocontrol strains of Pseudomonas that are able to effectively control pathogenic attack on crop plants.
  • Preferred biocontrol strains include the following strains of Pseudomonas fluorescens, which are described in detail in the examples below: CGA376146, CGA364473, CGA375258, CGA376148, CGA364476, CGA375260, CGA375259, CGA378584, CGA267pPhz, CGA364474, CGA364475, CGA366259, CGA376150, NOA402208, NOA402212, NOA402214, NOA402216, CGA267356/Phl, NOA409068, NOA413174, NOA413175, NOA413176, NOA413177, and NOA413178.
  • the biocontrol strains of the invention produce at least one antifungal substance that is capable of inhibiting a broad spectrum of plant pathogens such as Rhizoctonia and Pythium.
  • the biocontrol strains of the invention produce enhanced quantities of pyrrolnitrin; see, e.g., Table 1. As shown in Table 2, such strains have increased biocontrol properties and are able to aggressively compete in the plant rhizosphere.
  • Table 3 The genotypes of the biocontrol strains of the invention are summarized in Table 3 and deposit information for the biocontrol strains of the invention is given in Table 4.
  • the present invention is also intended to encompass pyrrolnitrin producing strains derived from the above-listed strains.
  • the present invention is also drawn to biocontrol compositions comprising the biocontrol strains of the invention in combination with a chemical fungicide such as a metalaxyl compound.
  • a chemical fungicide such as a metalaxyl compound.
  • SEQ ID NO:1 is the nucleotide sequence of the 11 kb EcoRI fragment containing the gacA gene and derived from the chromosome of Pseudomonas fluorescens strain CGA267356.
  • the coding sequences in this sequence include genes encoding: a methyltransferase (bases 210-1688) with homology to the cheR and frzF genes from E. coli and Myxococcus xanthus, respectively; a sensor kinase (bases 1906-3633) with homology to the rcsC, frzE and bvgS genes of E. coli, M.
  • tRNA bases 4616-4691 , complementary DNA strand
  • CDP-diacylglycerol-glycerol-3-phosphate-3-phosphatidyltransferase bases 4731-5318, complementary DNA strand
  • UVR exonuclease subunit C bases 5574-7397, complementary DNA strand
  • a response regulator/transcription activator gacA; bases 7400-8041 , complementary DNA strand
  • SEQ ID NO:2 is the nucleotide sequence of the native gacA regulatory gene.
  • SEQ ID NO:3 is the protein sequence encoded by the native gacA regulatory gene.
  • SEQ ID NO:4 is the nucleotide sequence of the ATG- gacA regulatory gene, wherein the first base in the coding sequence has been changed from the native thymidine (T) to an adenine (A) to create the more efficient ATG translation initiation codon.
  • SEQ ID NO:5 is the protein sequence encoded by the altered ATG- gacA regulatory gene.
  • SEQ ID NO:6 is the nucleotide sequence of the pyrrolnitrin gene cluster.
  • SEQ ID NO:7 is the nucleotide sequence of the tac promoter/rmB transcription terminator cassette.
  • SEQ ID NO:8 is the nucleotide sequence of the lemA gene.
  • SEQ ID NO:9 is the nucleotide sequence of the gac*3 regulatory gene, wherein the adenine (A) base at position 395 has been changed from the native adenine (A) to a guanine (G) so that codon 132 encodes an arginine residue instead of the usual glutamine.
  • SEQ ID NO:10 is the protein sequence encoded by the altered gac * 3 regulatory gene.
  • SEQ ID NO:11 is the nucleotide sequence of the phenazine gene cluster.
  • the present invention provides improved biocontrol strains of Pseudomonas that can be used to control pathogenic attack on crop plants. Such strains are able to aggressively compete in the plant rhizosphere as well as produce one or more antifungal substances that are effective against a broad spectrum of plant pathogenic fungi such as Rhizoctonia and Pythium.
  • the biocontrol strains of the present invention are important for several reasons.
  • Rhizoctonia such as Rhizoctonia solani are particularly pernicious plant pathogens.
  • the affected plants include beans, wheat, tomato and potato, in addition to cotton.
  • Pseudomonas fluorescens strain CGA267356 (also known as both MOCG134 and BL915) has been shown to be effective in controlling plant pathogenic fungi such as Rhizoctonia and Pythium.
  • Strain CGA267356 is one of the subjects of U.S. Patent No. 5,348,742.
  • Two mutants of CGA267356, strain s CGA321730 (a.k.a. MOCG134-8392) and CGA319115, have been constructed and shown to demonstrate even better biological control (biocontrol) of these phytopathogens.
  • CGA321730 and CGA319115 are the subject of U.S. Patent No. 5,496,547.
  • strain CGA267356 Two genes have been isolated from strain CGA267356 that encode proteins that regulate the synthesis of several antifungal compounds produced by the strain , including the antifungal metabolite pyrrolnitrin (prn). These are the /e A gene and gafA (a.k.a. gacA) gene that encode sensor kinase and response regulator proteins, respectively, which function as a typical two-component bacterial regulatory system. These genes and their use to activate biocontrol activity in biocontrol strains are described in U.S. Patent No. 5,670,350, which is hereby incorporated by reference in its entirety. In addition, U.S. Patent No. 5,639,949 describe a four gene cluster isolated from strain CGA267356 that encodes proteins that direct the biosynthesis of pyrrolnitrin.
  • the lemA and gacA regulatory genes and the pyrrolnitrin biosynthetic genes have been utilized to genetically modify parent Ps.
  • fluorescens strains such as CGA267356 and also mutant strains such as, for example , strains CGA321730 and CGA267356, to construct altered strains that demonstrate enhanced production of antifungal metabolites, i.e. pyrrolnitrin, and accordingly enhanced biocontrol activity.
  • genes from Pseudomonas aureofaciens strain 30-84 that are involved in the synthesis of the antifungal metabolite phenazine-1-carboxylic acid (PCA) have been utilized to genetically modify parent Ps. fluorescens strain CGA267356 to produce PCA, thereby improving the biocontrol activity of strain CGA267356.
  • plasmid pE11 is transformed into a transposon mutant of a wild-type P. fluorescens strain that has enhanced biocontrol activity over the wild-type strain resulting in a biocontrol strain according to the invention .
  • the first base in the coding sequence of the gacA gene is changed from a thymidine (T) to an adenine (A) to create the more efficient ATG translation initiation codon .
  • T thymidine
  • A adenine
  • This change can be created in vitro by PCR technology and the native gacA gene in the chromosome of a wild-type P. fluorescens strain can be replaced with the ATG/gacA gene by homologous gene replacement resulting in a biocontrol strain according to the invention .
  • a plasmid is introduced into a wild-type or mutant Pseudomonas strain, but preferably a P. fluorescens strain, comprising the pmABCD gene cluster with a strong constitutive bacterial promoter such as, for example, the tac promoter, upstream of the gene cluster and a suitable transcription terminator such as the trnB transcription terminator downstream of the pmABCD gene cluster.
  • a strong constitutive bacterial promoter such as, for example, the tac promoter
  • a suitable transcription terminator such as the trnB transcription terminator downstream of the pmABCD gene cluster.
  • the invention further provides a Pseudomonas strain transformed with a plasmid with the lemA and gacA genes derived from the chromosome of a wild-type strain.
  • a Pseudomonas strain which has been transformed to contain both the the pmABCD gene cluster and a plasmid with the lemA and gacA genes.
  • a single base change within the coding sequence of the native gacA gene is accomplished resulting in gacA genes that encode a GacA protein exhibiting a single amino acid change in the encoded GacA protein such that do no longer require phosphorylation by LemA in order to be active as a transcriptional activator.
  • Said gacA genes are isolated and used to replace the native gacA gene in wild- type and mutant Pseudomonas strains by perfect site replacement mediated through homologous recombination resulting in a biocontrol strain according to the invention .
  • a mutant gacA gene comprising a single base change in codon 132, which is CAG and encodes a glutamine residue in the native GacA protein.
  • the adenine base in this codon is changed to guanine to create a codon that encodes an arginine residue (CGG) in the altered strain.
  • CGG arginine residue
  • the LemA protein phosphorylates GacA and in the phosphorylated state it activates transcription of genes involved in the synthesis of antifungal compounds.
  • This single base change in the GacA protein renders it active irrespective of the kinase activity of the LemA protein.
  • the native promoter controlling expression of the chromosomal gacA gene is replaced with a strong constitutive bacterial promoter such as, for example, the tac promoter from E. coli and the thus modified gacA gene is introduction into a wild-type or mutant Peudomonas strain, optionally together with a plasmid comprising the pmABCD gene cluster.
  • a strong constitutive bacterial promoter such as, for example, the tac promoter from E. coli
  • the thus modified gacA gene is introduction into a wild-type or mutant Peudomonas strain, optionally together with a plasmid comprising the pmABCD gene cluster.
  • the newly inserted tac/gacA gene resulted in a higher level of expression of the gacA gene, compared to the low level of expression from the native gacA gene promoter.
  • the invention further provides a no-phenazine, pyrrolnitrin producing Pseudomonas strain which is transformed with a plasmid carrying DNA that contains 5 genes known to encode the pathway for the biosynthesis of the antifungal metabolite phenazine-1- carboxylic acid (PCA).
  • PCA antifungal metabolite phenazine-1- carboxylic acid
  • a plasmid is constructed comprising the lac promoter element from £. coli with the gacA gene such that expression of the gacA gene is regulated by the lac promoter.
  • This plasmid is transformed into a wild-type or mutant Pseudomonas strain resulting in a biocontrol strain according to the invention.
  • the tac promoter element from £. coli is operably linked with the pmABCD gene cluster and introduced in the chromosome of a wild- type or mutant Pseudomonas strain such that expression of the pmABCD genes is from a strong constitutive bacterial promoter such as, for example, the tac promoter.
  • a plasmid, but preferably plasmid pE11 containing the native gacA gene derived from P. fluorescens strain CGA267356 is introduced in said strain by conjugation.
  • the new strain is shown to produce higher amounts of the antifungal metabolite pyrrolnitrin and to provide greater biocontrol activity than the parent strain.
  • the invention also provides a 2-hexyl-5-propyl-resorcinol non-producing deletion mutant of a P. fluorescens strain. Said strain is transformed with a plasmid comprising the pmABCD gene cluster under the control of the tac promoter resulting in a biocontrol strain according to the invention.
  • a further embodiment of the invention pro vides a method for controlling or inhibiting the growth of a plant pathogenic fungus by applying the genetically engineered biocontrol strains of the invention to an environment in which the plant pathogenic fungus may grow. This can be to the plant/s or parts of the plant s (before or after harvest) or to the seeds (prior to planting) of the plant/s to be protected, or alternatively to soil in which the plant/s to be protected are growing or will grow.
  • the biocontrol strains are applied in an effective amount; that is, in an amount sufficient to control or inhibit the pathogen.
  • the rate of application may vary according to the crop to be protected, the efficacy of the biocontrol strain, the pathogen to be controlled, and the severity of the disease pressure.
  • the rate of application is about 1.3 x 10 5 cfu/cm to about 1.3 x 10 10 cfu/cm, specifically about 1.3 x 10 6 cfu/cm to about 1.3 x 10 9 cfu/cm, more specifically about 1.3 x 10 7 cfu/cm to about 1.3 x 10 8 cfu/cm.
  • a more particular embodiment of the present invention provides methods of inhibiting the growth of Rhizoctonia and Pythium by applying the biocontrol strains of the invention to environments in which the plant pathogenic fungi may grow. This can be to the plant/s or parts of the plant/s (before or after harvest) or to the seeds (prior to planting) of the plant/s to be protected, or alternatively to soil in which the plant/s to be protected are growing or will grow. As noted above, the rate of application varies depending on various factors.
  • the general rate of application is about 1.3 x 10 5 cfu/cm to about 5 x 10 9 cfu/cm, specifically about 1.3 x 10 6 cfu/cm to about 1.3 x 10 9 cfu/cm more specifically about 1.3 x 10 7 cfu/cm to about 1.3 x 10 8 cfu/cm.
  • the recombinant biocontrol strains of the present invention may be used in any manner known in the art, including coating seeds with an effective amount of the biocontrol strains, in furrow application of the biocontrol strains directly into the soil, in foliar application, and in post-harvest disease control.
  • Such methods are well known in the art and are described, for example, in U.S. Patent No. 5,348,742 and in the published European Application EP 0 472 494 A2, which is hereby incorporated by reference.
  • the strains of this application can also be mixed in formulation with known pesticides in a manner described in WO 94/10845, which disclosure is herein incorporated by reference.
  • Strain CGA376146 (a.k.a. MON0571) was constructed by introducing plasmid pE11 into P. fluorescens strain CGA321730 (a.k.a. MOCG-0134-8392) by conjugation. Strain CGA321730 is described in U.S. Patent No. 5,496,547 and is a transposon mutant of wild- type P. fluorescens strain CGA267356 (U.S. Pat. No. 5,348,742) that has enhanced biocontrol activity over the wild-type strain. Strain CGA267356 has been deposited with the ATCC and assigned ATCC accession number 55169. Strain CGA321730 has been deposited with the NRRL and assigned accession number NRRL B-21173.
  • Plasmid pE11 (Patent No. 5,670,350) was constructed by ligating the 1 1 kilobase (kb) EcoRI fragment derived from the chromosome of strain CGA267356 into the broad host range plasmid vector pRK290 (Dittaera/.,P/oc. Na ⁇ .Acad. S ⁇ . USA 77:7347-7351 (1980). Plasmid pE11 has been deposited with ATCC and has been assigned ATCC accession number 40869.
  • the 11-kb EcoRI fragment contains the gacA (gafA) gene that encodes a response regulator protein known to regulate the synthesis of antifungal compounds.
  • This 11-kb EcoRI fragment is described in U.S. Patent No.5,670,350 and is set forth herein as SEQ ID NO. .
  • the plasmid vector, pRK290, used in constructing pE11 is derived from native Pseudomonas plasmids. It is mobilizable but not self-transmissible by conjugation and it carries a tetracycline resistance gene (DittaetaL, 1980).
  • New strain CGA376146 has been shown to produce higher amounts of the antifungal metabolite pyrrolnitrin (Table 1 ) and to have higher biocontrol activity (Table 2) compared to the related strains CGA267356 and CGA321730.
  • New strain CGA376146 was deposited with the NRRL on September 5, 1997, and assigned accession no. NRRL B- 21811.
  • P. fluorescens strain CGA364473 (a.k.a. MON0517) was derived from parent P. fluorescens strain CGA267356 by changing a single base in the chromosome of the parent strain.
  • the native gacA regulatory gene begins with the unusual TTG translation initiation codon (SEQ ID NO:2). All proteins in nature are synthesized with methionine as the first amino acid on the amino terminus and ATG is the only codon that encodes methionine. Therefore, the normal translation initiation codon for most genes is ATG. Alternate translation initiation codons GTG and TTG will also result in the incorporation of methionine as the first amino acid since methionine must always be the first amino acid in a newly synthesized protein, but they usually cause a reduction in the efficiency of translation. As a result, fewer protein molecules are made from the same amount of messenger RNA.
  • strain CGA364473 To create strain CGA364473, the first base in the coding sequence of the gacA gene was changed from a thymidine (T) to an adenine (A) to create the more efficient ATG translation initiation codon (SEQ ID NO:4).
  • T thymidine
  • A adenine
  • SEQ ID NO:4 This change was created in vitro by PCR technology and the native gacA gene in the chromosome of strain CGA267356 was replaced with the ATG/gacA gene by homologous gene replacement.
  • the amino acid sequence of the GacA protein encoded by this altered gacA gene (SEQ ID NO:5) is identical to that encoded by the native gene (SEQ ID NO:3), but translation should be more efficient, resulting in synthesis of higher amounts of the protein.
  • New strain CGA364473 has been shown to produce higher amounts of the antifungal metabolite pyrrolnitrin (Table 1 ) and to have higher biocontrol activity (Table 2) compared to strain CGA267356.
  • New strain CGA364473 was deposited with the NRRL on September 5, 1997, and assigned accession no. NRRL B-21812.
  • Strain CGA375258 (a.k.a. MON0568) was created by introducing plasmid pPrn into the parent P. fluorescens strain CGA267356. Plasmid pPrn was constructed by cloning a 6.2 kb Xba ⁇ /Not gene fragment from plasmid pCIB169 (which was derived from P. fluorescens strain CGA267356) into the expression vector pKK223-3 , as described in Examples 7-11 of U.S. Patent No. 5,639,949 and Application Serial No. 08/729,214. (See SEQ ID NO:6). Plasmid pCIB169 has been deposited with the NRRL and assigned accession number NRRL B-21256.
  • the 6.2 kb XbaUNott gene fragment of SEQ ID NO:6 contains the pmABCD gene cluster that encodes genes for the biosynthesis of pyrrolnitrin as described in Patent No. 5,639,949 and Serial No. 08/729,214.
  • a 6.9 kb ⁇ g/ll fragment containing the pmABCD gene cluster with the tac promoter (see SEQ ID NO:7) upstream of the gene cluster and the rrnB transcription terminator (SEQ ID NO:7) derived from plasmid pKK223-3 downstream of the cluster was subsequently cloned into the Sg/ll site of plasmid pRK290 to create plasmid pPrn.
  • the tac promoter is a small DNA fragment (less than 100 bases) derived from E. coli, which is known to be a regulatory e iement or promoter (Amann, etal, Gene 25:167-178 (1983)) that does not itself encode a protein product.
  • SEQ ID NO:7 presents the sequence of the SssHII DNA fragment containing the tac promoter and the rrnB transcription terminator derived from plasmid pKK223-3.
  • the tac promoter is known to be highly expressed in a constitutive manner in Pseudomonas. Its use with the pm genes causes constitutive, high-level expression of these genes.
  • the promoter for the pyrrolnitrin genes is regulated by the gacA gene product; accordingly, the pyrrolnitrin genes are expressed in the native strain only in the stationary phase of growth.
  • New strain CGA375258 has been shown to produce higher amounts of the antifungal metabolite pyrrolnitrin (Table 1 ) and to have higher biocontrol activity (Table 2) compared to the related strain CGA267356.
  • New strain CGA375258 was deposited with the NRRL on September 5, 1997, and assigned accession no. NRRL B-21813.
  • strain CGA376148 contains the plasmid pPrn (Example 3) that carries the tac promoter fused to the pmABCD gene cluster.
  • New strain CGA376148 has been shown to produce higher amounts of the antifungal metabolite pyrrolnitrin (Table 1 ) and to have higher biocontrol activity (Table 2) compared to the related strain CGA267356.
  • New strain CGA376148 was deposited with the NRRL on September 5, 1997, and assigned accession no. NRRL B-21814.
  • Strain CGA364476 (a.k.a. MONO520) is the same as wild-type strain CGA267356, except that strain CGA364476 also contains a plasmid with the lemA and gacA genes derived from the chromosome of the wild-type strain CGA267356.
  • the plasmid containing the lemA and gacA genes, pLem/Gac was constructed as follows:
  • the plasmid pCIB146 (Examples 20 and 21 and Figure 4 of Patent No. 5,670,350) contains about 25-kb of chromosomal DNA from strain CGA267356.
  • the lemA gene (see SEQ ID NO:8) has been described and shown to be located in this DNA.
  • the subcloned DNA in pCIB146 is flanked on each side by Not ⁇ and EcoRI sites. An approximately 16-kb Hind ⁇ INot ⁇ fragment from pCIB146, which contains the lemA gene, was excised from pCIB146.
  • the Not ⁇ end was converted to a Hind ⁇ site to facilitate cloning of the fragment into the unique Hind ⁇ fragment of plasmid pE11 (Example 1) , which contains the gacA gene. Insertion of the 16-kb /emA-containing Hind ⁇ fragment into the Hind ⁇ site of pE11 did not disrupt the function of the gacA gene, because the H/ndlll site is not within its coding sequence.
  • New strain CGA364476 has been shown to produce higher amounts of the antifungal metabolite pyrrolnitrin (Table 1 ) and to have higher biocontrol activity (Table 2) compared to the related strain CGA267356.
  • New strain CGA364476 was deposited with the NRRL on September 5, 1997, and assigned accession no. NRRL B-21815.
  • Strain CGA375260 differs from the parent strain CGA267356 by a single base change within the coding sequence of the native gacA gene (SEQ ID NO:2). This modification was generated by introducing the native gacA gene into the hypermutagenic E. coli strain XL1-Red (from Stratagene, Inc.). The plasmid was recovered and introduced into a lemA mutant of strain CGA267356 that also contained lacZY genes inserted into an unknown chromosomal gene whose expression is regulated by LemA and GacA.
  • the GacA protein (SEQ ID NO: 10) in this strain has an arginine at amino acid 132 instead of the usual glutamine.
  • this strain is identical to the parent strain.
  • the LemA protein phosphorylates GacA and in the phosphorylated state it activates transcription of genes involved in the synthesis of antifungal compounds. This single base change in the GacA protein renders it active irrespective of the kinase activity of the LemA protein.
  • New strain CGA375260 was deposited with the NRRL on September 5, 1997, and assigned accession no. NRRL B-21816.
  • Strain CGA375259 (a.k.a. MON0569) was derived from strain CGA267356 by replacement of the native promoter controlling expression of the chromosomal gacA gene with the tac promoter from E. coli (Example 3) and introduction of plasmid pPrn (Example 3).
  • the promoter of the gacA gene was replaced with the tac promoter as follows: A unique Nru ⁇ site in the 2-kb Xho ⁇ gacA gene-containing fragment of pCIB137 (Examples 6 and 7 of Patent No. 5,670,350) located 12-bp upstream of the translation start site of the gacA gene was modified by PCR to change it to a SamHI site. Plasmid pCIB137 has been deposited with the NRRL and assigned accession number NRRL B-18981. A second SamHI site was inserted immediately 5' to the gacA translation start site using PCR. This created a small SamHI fragment immediately preceding the gacA coding sequence.
  • This short SamHI fragment was excised and the DNA was religated to create a new SamHI site.
  • the tac promoter was excised from plasmid pKK223-3 (SEQ ID NO:7) as a Sg/ll/SamHI fragment and cloned in the appropriate orientation into the new SamHI site 5' to the beginning of the gacA gene, which was created by the excision of the above short SamHI fragment.
  • This tac promoter/gacA gene (tac/gacA) fragment was excised as an Xho ⁇ fragment and was used to replace the native gacA gene on the 2-kb Xho ⁇ site in a plasmid containing the Hind ⁇ WEcoR ⁇ fragment from pE11.
  • This plasmid was introduced into a gacA deletion mutant of strain CGA267356 (Example 9 of Patent No. 5,670,350). Perfect replacement clones were generated (Example 9 of Patent No. 5,670,350) and selected by restoration of the wild-type colony morphology.
  • the newly inserted tac/gacA gene resulted in a higher level of expression of the gacA gene, compared to the low level of expression from the native gacA gene promoter.
  • Plasmid pPrn described in Example 3 above was subsequently introduced into the strain containing the tac/gacA gene to make strain CGA375259.
  • New strain CGA375259 has been shown to produce higher amounts of the antifungal metabolite pyrrolnitrin (Table 1 ) and to have higher biocontrol activity (Table 2) compared to the related strain CGA267356.
  • New strain CGA375259 was deposited with the NRRL on September 5, 1997, and assigned accession no. NRRL B-21817.
  • Strain CGA378584 (a.k.a. MON0591 ) contains the tac/pmABCD gene cluster described in Example 3 in the chromosome and it also contains plasmid pLem/Gac described in Example 5.
  • the tac/pmABCD genes were inserted in the chromosome of strain CGA267356 by modification of the £. coli cloning vector pKK223-3 by adding a kanamycin resistance gene derived from plasmid pUC4K (Pharmacea) into the Pst ⁇ site of the multiple cloning site and by removing the EcoRI, Nott, and SamHI sites of pKK223-3.
  • a 16-kb Kpn ⁇ gene fragment derived from pCIB169 ( Figure 4 of Patent No. 5,639,949) was cloned into the modified pKK223-3. Plasmid pCIB169 has been deposited with the NRRL and assigned accession number NRRL B-21256.
  • the pmABCD genes were deleted by digestion of the plasmid with EcoRI and Noti, conversion of the EcoRI and ⁇ /ofl ends to SamHI by fill-in, linkering, and religation.
  • the 6.9- kb Sg/ll fragment (Example 3), which contains the tac promoter/ pmABCD/ rmB terminator construction, was ligated into the SamHI site, thus introducing these modified genes into the plasmid.
  • This plasmid was introduced into strain CGA267356 by conjugation and the native pmABCD gene cluster was replaced with the tac promoter/pmABCD/tmB terminator construct by homologous recombination.
  • Plasmid pLem/Gac (Example 5) was introduced into the strain with the chromosomally located tac promoter/pmABCD/tmB terminator to create strain CGA378584.
  • New strain CGA378584 has been shown to produce higher amounts of the antifungal metabolite pyrrolnitrin (Table 1 ) and to have higher biocontrol activity (Table 2) compared to the related strain CGA267356 .
  • New strain CGA378584 was deposited with the NRRL on September 5, 1997, and assigned accession no. NRRL B-21818.
  • strain CGA267pPhz contains a plasmid carrying DNA from Pseudomonas aureofaciens strain 30-84 that contains 5 genes known to encode the pathway for the biosynthesis of the antifungal metabolite phenazine-1-carboxylic acid (PCA).
  • Plasmid pUCP26 is an E. coli/Pseudomonas shuttle plasmid that has a lac promoter flanking the multiple cloning site.
  • the EcoRI and Hind ⁇ sites are oriented with the lac promoter such that the EcoRI site is closer to the promoter. Therefore, cloning of the EcoRI/H/ ⁇ dlll p ⁇ zFABCD gene fragment, in which the phz genes are cotranscribed on a single operon oriented in the EcoRI to HindlW direction into plasmid pUCP26, results in the proper juxtaposition of the lac promoter and phz gene cluster to cause expression of these genes from that promoter.
  • New strain CGA267pPhz has been shown to produce PCA, an antifungal metabolite not normally produced by parent strain CGA267356. It also produces the metabolites normally produced by the parent strain, including pyrrolnitrin. New strain CGA267pPhz was deposited with the NRRL on September 5, 1997, and assigned accession no. NRRL B- 21819.
  • Strain CGA364474 (a.k.a. MON0518) was constructed by juxtaposition of the lac promoter element from E. coli (de BoeretaL, Free. Nat. Acad Set. USA 8021-25 (1983)) with the gacA gene (SEQ ID NO:2) residing in the chromosome of P. fluorescens strain CGA267356 (U.S. Patent No. 5,348,742) such that expression of the gacA gene is regulated by the lac promoter.
  • the lac promoter is a small DNA fragment that is known to promote gene expression and does not itself encode a protein product. It provides strong, constitutive expression of genes in Pseudomonas.
  • lac promoter and the gacA gene were fused precisely by overlapping PCR and the lac/gacA promoter/gene DNA fragment replaced the native gacA gene in the chromosome of strain CGA267356 by homologous recombination.
  • Strain CGA364474 has been shown to produce higher amounts of the antifungal metabolite pyrrolnitrin (Table 1 ) and to provide higher biocontrol activity (Table 2) than parent strain CGA267356. New strain CGA364474 was deposited with the NRRL on November 20, 1997, and assigned accession no. NRRL B-21887.
  • P. fluorescens strain CGA364475 (a.k.a. MON0519) was derived from the parent P. fluorescens strain CGA267356 by juxtaposition of the tac promoter element from E. coli (Example 3, SEQ ID NO:7) with the pmABCD gene cluster (U.S. Patent No. 5,639,949) in the chromosome such that expression of the pmABCD genes is from the tac promoter.
  • the pmABCD genes are expressed from the strong, constitutive tac promoter rather than the weaker, regulated native pm gene promoter.
  • this strain produces more of the antifungal metabolite pyrrolnitrin (Table 1 ) and provides greater biocontrol activity (Table 2) than parent strain CGA267356.
  • New strain CGA364475 was deposited with the NRRL on November 20, 1997, and assigned accession no. NRRL B- 21888.
  • Strain CGA366259 (a.k.a. MON0524) was created by introducing plasmid pE11 (Example 1 ) containing the native gacA gene derived from P. fluorescens strain CGA267356 into P. fluorescens strain CGA364475 (Example 11) by conjugation. New strain CGA366259 has been shown to produce higher amounts of the antifungal metabolite pyrrolnitrin (Table 1 ) and to provide greater biocontrol activity (Table 2) than parent strain CGA267356. New strain CGA366259 was deposited with the NRRL on November 20, 1997, and assigned accession no. NRRL B-21889. EXAMPLE 13 Construction of Strain CGA376150 (MON0575) (CGA267355, pE11 )
  • Strain CGA376150 (a.k.a. MON0575) was constructed by introducing plasmid pE11 (Example 1 ) into the wild-type P. fluorescens strain CGA267355 by conjugation. Strain CGA267355 was isolated from soil in Texas. It normally does not produce the antifungal metabolites pyrrolnitrin and 2-hexyl-5-propyl-resorcinol , or the hydrolytic enzyme chitinase.
  • strain CGA376150 was demonstrated to produce pyrrolnitrin (Table 1), 2-hexyl-5-propyl- resorcinol, and chitinase and was shown to be an effective biocontrol agent (Table 2).
  • New strain CGA376150 was deposited with the NRRL on November 20, 1997, and assigned accession no. NRRL B-21890.
  • Strain NOA402208 (a.k.a. MONO630) was constructed by introducing the plasmid pPrn containing the pmABCD gene cluster under the control of the tac promoter (Example 3) into P. fluorescens strain CGA375260 (Example 6) by conjugation.
  • Strain CGA375260 differs from the parent strain CGA267356 by a single base change in the gacA coding sequence (SEQ ID NO:9) that renders GacA (SEQ ID NO:10), the protein product of the modified gacA gene and an activator of transcription, active irrespective of phosphorylation by the LemA protein.
  • strain NOA402208 produces more pyrrolnitrin (Table 1) and has greater biocontrol activity (Table 2) than parent strain CGA267356.
  • New strain NOA402208 was deposited with the NRRL on November 20, 1997, and assigned accession no. NRRL B-21891.
  • Strain NOA402210 (a.k.a. MON0632) was constructed from P. fluorescens strain CGA267356 (U.S. Patent No. 5,348,742) by deletion of a region of the chromosome that resulted in no production of the antimicrobial metabolite 2-hexyl-5-propyl-resorcinol and introduction of the plasmid pPrn by conjugation.
  • P. fluorescens strain CGA319115 is a transposon mutant of the parent strain CGA267356 that is incapable of the production of 2-hexyl-5-propyl-resorcinol (U.S. Patent No. 5,496,547) and that provides greater biocontrol activity compared to strain CGA267356.
  • a cosmid clone, BL3610, from a gene library of DNA from strain CGA267356 was found that restores production of 2-hexyl-5-propyl-resorcinol to strain CGA319115.
  • An in vivo marker exchange was performed with cosmid clone BL3610 in strain CGA319115 in order to rescue the transposon and the flanking DNA.
  • cosmid clone BL3610Tn containing the mutagenized genomic DNA from strain CGA319115 with the transposon was thus isolated.
  • a 6.5 kilobase pair (kb) EcoRI DNA fragment from cosmid BL3610 that corresponded to the region in cosmid BL3610Tn that contained the transposon insertion was cloned into plasmid pBluescript II (Pharmacea, Inc.) to create plasmid pBL3632.
  • Strain NOA402210 produces more pyrrolnitrin (Table 1) than the parent strain CGA267356 and it provides greater biocontrol activity (Table 2) compared to the parent strain.
  • Strain NOA402210 was deposited with the NRRL on November 20, 1997, and assigned accession no. NRRL B-21901.
  • Strain NOA402212 (a.k.a. MON0634) was constructed in a manner identical to that described for strain NOA40221 0 in Example 15 above, except that the strain used as the starting strain for NOA402212 was P. fluorescens strain CGA375260 (Example 6) instead of strain CGA267356.
  • Strain NOA402212 was created by introducing the deletion of the 200 bp Sc/l fragment into the chromosome of P. fluorescens strain CGA375260 to create strain NOA402211 and by the subsequent introduction of the plasmid pPrn containing the pmABCD gene cluster under the control of the tac promoter. This strain produces more pyrrolnitrin (Table 1) than either the parent strain CGA267356 or strain NOA402208 and it provides greater biocontrol activity (Table 2) compared to the parent strain.
  • Strain NOA402212 was deposited with the NRRL on November 20, 1997, and assigned accession no. NRRL B-21892.
  • Strain NOA402214 (a.k.a. MON0636) was constructed in a manner identical to that described for strain NOA40221 0 in Example 15 above, except that the strain used as the starting strain for NOA402214 was P. fluorescens strain CGA364473 (Example 2) instead of strain CGA267356.
  • Strain CGA364473 is identical to strain CGA267356 except for a single base change in the coding sequence of the gacA gene.
  • the gacA gene was noted to have the unusual TTG translation start codon.
  • the first base in the coding sequence of the gacA gene was changed to an adenine (A), thus creating the normal ATG translation start codon ( SEQ ID NO:4).
  • Strain CGA364473 was further modified in the same manner as strain NOA402212 to contain the deletion of the 200 base pair Sc/l fragment, creating strain NOA402213 , which does not produce 2-hexyl-5-propyl-resorcinol.
  • Strain NOA402214 was created by introduction of the plasmid pPrn containing the pmABCD genes under the control of the tac promoter by conjugation into strain NOA402213.
  • Strain NOA402214 produces higher amounts of pyrrolnitrin (Table 1) and provides greater biocontrol activity (Table 2) than parent strain CGA267356.
  • Strain NOA402214 was deposited with the NRRL on November 20, 1997, and assigned accession no. NRRL B-21893.
  • Strain NOA402216 (a.k.a. M0N0638) contains a chromosomal gacA gene with an ATG translation start codon as in Example 17 that is expressed from the lac promoter of E. coli. It also contains a chromosomal deletion of the 200 base pair Sc/l fragment that results in a 2-hexyl-5-propyl-resorcinol non-producing phenotype. Finally, it contains the plasmid pPrn described in Example 14, which contains the pmABCD gene cluster under regulation of the tac promoter.
  • the ATG-gacA gene was fused to the lac promoter and integrated into the chromosome of P. fluorescens strain CGA267356 essentially as described in Example 7, except that the lac promoter was used instead of the tac promoter.
  • the resulting strain containing the lac/ATG-gacA gene in its chromosome was converted to a 2-hexyl-5-propyl- resorcinol non-producing phenotype by the same method described for the construction of strain NOA402212 (Example 15) to create strain NOA402215.
  • strain NOA402216 was constructed by introduction of the plasmid pPrn into strain NOA402215.
  • Strain NOA402216 produces more pyrrolnitrin (Table 1) and provides greater biocontrol activity (Table 2) than parent strain CGA267356.
  • Strain NOA402216 was deposited with the NRRL on November 20, 1997, and assigned accession no. NRRL B- 21894.
  • Strain NOA409063 (a.k.a. MON0686) was constructed from the parent P. fluorescens strain CGA267356 by the introduction of a plasmid containing both the gac*3 and pmABCD genes. Plasmid pGac * 3/Pm was constructed by cloning the pmABCD gene cluster under the control of the tac promoter as a 6.9 kb Sg ll fragment (Example 3) into the unique Sg/ll site of the broad host-range plasmid pVK101 (Knauf, V. and Nester, E.
  • Strain NOA409068 (a.k.a. MON0691) was constructed in a manner identical to that described for strain CGA375258 in Example 3 above, except that plasmid pKT231 was used in strain NOA409068 to maintain the exogenous DNA instead of the plasmid pRK290 used in strain CGA375258.
  • Strain NOA409068 was created by introducing plasmid pKT-Prn into parent P. fluorescens strain CGA267356 by conjugation. Plasmid pKT-Prn was constructed by cloning the 6.9 kb Sg/ll fragment described in Example 3 containing the pmABCD gene cluster from strain CGA267356 with the tac promoter and rmB transcription terminator, into the broad host-range plasmid pKT231 (P ⁇ hler, Vectors for Gram-negative Bacteria. 1985. Elsevier Scientific Publishers).
  • Strain NOA409068 produces more pyrrolnitrin (Table 1 ) and provides greater biocontrol activity (Table 2) than parent strain CGA267356.
  • Strain NOA409068 was deposited with the NRRL on November 20, 1997, and assigned accession no. NRRL B- 21895.
  • Strain NOA413174 (a.k.a. MONO706) was created by introduction of plasmid pKT- Prn (Example 20) by conjugation into the P. fluorescens 2-hexyl-5-propyl-resorcinol non- producing strain NOA402209 described in Example 15. Strain NOA413174 produces more pyrrolnitrin (Table 1 ) and provides greater biocontrol activity (Table 2) compared to parent strain CGA267356. Strain NOA413174 was deposited with the NRRL on November 20, 1997, and assigned accession no. NRRL B-21896.
  • EXAMPLE 22 Construction of Strain NOA413175 (MONO707) (gac*3, pKT-Prn)
  • Strain NOA413175 (a.k.a. MONO707) was created by introduction of plasmid pKT- Prn (Example 20) by conjugation into the P. fluorescens strain CGA375260 (Example 6). Strain NOA413175 produces more pyrrolnitrin (Table 1) and provides greater biocontrol activity (Table 2) than parent strain CGA267356. Strain NOA413175 was deposited with the NRRL on November 20, 1997, and assigned accession no. NRRL B-21897. EXAMPLE 23 Construction of Strain NOA413176 (MONO708) (gac * 3, res ' , pKT-Prn)
  • Strain NOA413176 (a.k.a. MONO708) was created by introduction of plasmid pKT- Prn (Example 20) by conjugation into P. fluorescens strain NOA402211 (described in Example 16). Strain NOA413176 produces more pyrrolnitrin (Table 1) and provides greater biocontrol activity (Table 2) than parent strain CGA267356. Strain NOA413176 was deposited with the NRRL on November 20, 1997, and assigned accession no. NRRL B- 21898.
  • EXAMPLE 24 Construction of Strain NOA413177 (MONO709) (ATG-gacA, res ' , pKT-Prn)
  • Strain NOA413177 (a.k.a. MONO709) was created by introduction of plasmid pKT- Prn by conjugation into P. fluorescens strain NOA402213 (described in Example 17). Strain NOA413177 produces more pyrrolnitrin (Table 1) and provides greater biocontrol activity (Table 2) than parent strain CGA267356. Strain NOA413177 was deposited with the NRRL on November 20, 1997, and assigned accession no. NRRL B-21899.
  • Strain NOA413178 (a.k.a. MONO710) was created by introduction of plasmid pKT- Prn by conjugation into P. fluorescens strain NOA402215 (described in Example 18). Strain NOA413178 produces more pyrrolnitrin (Table 1) and provides greater biocontrol activity (Table 2) than parent strain CGA267356. Strain NOA413178 was deposited with the NRRL on November 20, 1997, and assigned accession no. NRRL B-21900.
  • EXAMPLE 26 Cultivation of Bacteria and Fungi for Screening Assays a. Cultivation of bacteria The bacterial strains are stored in 20% glycerol at -80 9 C prior to use. One loop from the stored culture is suspended in 5 ml Luria Broth (LB: 10 g Bacto-Tryptone, Difco; 5 g yeast extract, Oxoid; 0.25 g MgS0 4 H 2 0; 8 g NaCI; and 1 L distilled water; pH 7) and shaken at 150 rpm and 25 9 C overnight. 100 ml LB is inoculated with 1 ml of the preculture and incubated under the same conditions.
  • LB Luria Broth
  • 10 ml of the last culture are centrifuged (10 min at 10,000 rpm), and the pellet is resuspended in 200 ml saline (0.8% NaCI) giving a concentration of approximately 10 8 cfu/ml.
  • a dilution series (10° to 10 "8 , 20 ⁇ l in 180 ⁇ l) is prepared in microtiter plate and drops of 10 ⁇ l are spotted onto Luria Agar (LB with 1.5% Bacto-Agar, Difco) with an Eppendorf pipette. The cfu are counted after 24 hrs incubation at 28 9 C.
  • Antibiotics may be added if required for selection of bacteria: tetracycline - 15 ⁇ g/ml; kanamycin ⁇ 50 ⁇ g/ml.
  • Cultivation of Rhizoctonia solani Rhizoctonia solani is grown on Potato Dextrose Agar (PDA, Difco) pH 5.6 in a petri dish. A 300 ml Erlenmeyer flask with 25 g millet and 50 ml distilled water is autoclaved and incubated with one agar plug (5mm diameter) from a PDA culture of R. solani.
  • Preparation of bacterial cultures All bacteria cultures are cultured in Luria broth for 2 days at 28° C. Bacterial cells are collected by centrifugation and resuspended in water to 10 9 or 10 8 bacterial cells/ml. 10 ml of each suspension is used to drench a pot containing 50 ml soil, resulting in 2 x 10 8 (high rate, CGA267356 only) or 2 x 10 7 cells/ml soil (all strains).
  • Rhizoctonia solani is grown on twice-autoclaved millet seed until fully colonized, then air dried for several days. Dried inoculum is ground to a fine powder for use in all assays except the poinsettia assays, in which whole colonized millet seeds are used. A large supply of inoculum is stored at room temperature and used for several months. Pythium aphanidermatum is inoculated on twice-autoclaved millet seed and grown for one week. The colonized millet seed is air dried for 2 hours, then used immediately. Pym/ ' um-infested millet seed is prepared weekly.
  • Rhizoctonia so/an/ ' -cucumber One cucumber seed is planted per pot containing a standard commercially available peat/bark type potting soil. The bacterial suspension is drenched on each pot and Rhizoctonia inoculum is broadcast over the surface of the soil. Each treatment in an experiment contains 5 sets of 12 plants which are randomized and placed in greenhouse under automatic sprinklers. Each experiment is repeated a minimum of three times. Stand counts are recorded at 1 and 2 weeks after planting and compared to uninfested and untreated healthy controls and infested and untreated diseased controls.
  • a single hole is drilled in the center of each pot and Rhizoctonia inoculum is broadcast over the surface of the soil and into the hole.
  • One commercially purchased impatiens seedling plug is transplanted into the hole of each pot and bacteria suspension is applied as a drench.
  • Each treatment in an experiment contains 5 sets of 12 plants which are randomized and placed in greenhouse under automatic sprinklers. Each experiment is repeated a minimum of three times. Stand counts are recorded at 1 and 2 weeks.
  • the bacterial suspension is drenched on strips of ten Oasis rooting cubes (the cell suspensions were adjusted so that each 50ml cube was drenched with 40ml of suspension), resulting in 2 x 10 8 or 2 x 10 7 cells/ml of cube, and one commercially purchased poinsettia cutting is inserted into each cube in the normal method for rooting.
  • Five Rhizoctonia -infested millet seeds are placed in the middle of the strip (between plants 5 and 6), and the strips are placed in the greenhouse under automatic sprinklers.
  • Each treatment in an experiment contains 4 rooting strips with 10 cuttings each and each experiment is repeated a minimum of three times. Stand counts are recorded at 10 days and 21 days after planting.
  • Pathosystem Pythium aphanidermatum-c c mber One cucumber seed is planted per pot and the bacterial suspension is drenched on top of each pot. Pythium inoculum is broadcast over the surface of the soil and replicates are randomized and placed in the greenhouse under automatic sprinklers. Each treatment in an experiment contains 5 sets of 12 plants and each experiment is repeated a minimum of three times. Stand counts are recorded at 1 and 2 weeks .
  • Active antifungal metabolites such as pyrrolnitrin (prn) can be extracted from the growth medium of bacterial strains that produce inhibitory antibiotics. For example, using strain CGA376146, this can be accomplished by extraction of the growth medium with 80% acetone followed by removal of the acetone by evaporation and a second extraction with diethyl ether. The diethyl ether is removed by evaporation and the dried extract is resuspended in a small volume of methanol.
  • the antifungal metabolites can be extracted with methanol using conventional methods. Small aliquots of the antibiotic extract applied to small sterile filter paper discs placed on an agar plate will inhibit the growth of Rhizoctonia solani, indicating the presence of the active antibiotic compound.
  • the biocontrol strains of the invention are each applied to non-sterile soil as a drench at 2 x 10 8 cfu/ml soil, while metalaxyl fungicide is either drenched (Ridomil at 0.02, 0.5, or 2 ppm) or coated onto seeds (Apron at 35 g a.i./100 kg seed).
  • Pythium aphanidermatum is introduced as an oospore suspension (1400 spores/ml soil).
  • Rhizoctonia solani is introduced as a pelleted millet powder (5 mg in the center of each pot). After incubation for 19 days in the greenhouse, the hypocotyls of cotton seedlings are rated for disease on an observation scale.
  • Formulations of antifungal compositions containing as the active ingredient th e antifungal metabolites that are produced by the biocontrol strains of the invention and that are inhibitory to the growth of Rhizoctonia and Pythium are produced according to Examples 10 and 11 in U.S. Patent No. 5,348,742. These formulations include emulsifiable concentrates, solutions, granulates, dusts, wettable powders, extruder granulates, coated granulates, and suspension concentrates.
  • the antifungal compositions may be used to control or inhibit the growth of a plant pathogenic fungus by applying an effective amount of the biocontrol composition to an environment in which the fungus may grow, to a plant or plant part, and/or to seed.
  • Cultures of the biocontrol strains are stored in 20% glycerol at -80 s C.
  • One loop from the stored culture is suspended in 5 ml Luria Broth (LB: 10 g Bacto-Typtone, Difco; 5 g yeast extract, Oxold; 0.25 g MgS0 4 H 2 0; 8 g NaCI; and 1 L distilled water; pH 7) and shaken at 150 rpm and 25 s C for 24 hrs.
  • 100 ml LB is inoculated with 1 ml of the preculture and incubated under the same conditions.
  • the culture is centhfuged for 10 minutes at 10,000 rpm, and the pellet is resuspended in saiine (0.8% NaCI) and adjusted to 3 x 109 cfu/ml (OD2).
  • saiine 0.8% NaCI
  • OD2 3 x 109 cfu/ml
  • a dilution series (10 9 to 10 " ⁇ 20 ⁇ l in 180 ⁇ l) is prepared in a microtiter plate and drops of 10 ⁇ l are spotted onto Luria Agar (LB with 1.5% Bacto-Agar, Difco) with an Eppendorf pipette. The cfu are counted after 24 hrs incubation at 28 s C.
  • Rhizoctonia and Pythium are prepared for inoculation as in Examples 10 and 11 above.
  • Emergence is recorded at 10 days after planting to assess pre-emergence damping off. Stands are recorded at 21 days and 28 days after planting to assess post-emergence damping-off.
  • Plasmid pRK290 carrying an 11 kb EcoRI fragment with the native gacA gene.
  • Plasmid pRK290 carrying the pmABCD gene cluster under control of the tac promoter.
  • c 2-hexyl-5-propyl-resorcinol non-producing phenotype from a chromosomal deletion.
  • d Plasmid pKT231 carrying the pmABCD gene cluster under control of the tac promoter.
  • MOLECULE TYPE DNA (genomic)
  • CDP-diacylglycerol-glycerol-3-phosphate-3-phosphatidyltransfere se has homology to pgsA.
  • gacA (aka gafA) has homology to the uvrY and gacA genes of E. coli and Ps. fluorescens, respectively.
  • CTGCGTOGCC T3CCGACGAC CAAGGCGQCC GCCCCTGCCC TCAAQQCGGT CAATACCATT 2640
  • GQCAATOCCA GGAACAACAG TTACGACCTG ATCATTTCAG ACATCQQCAT Q.CGATTATG 3420
  • GCGQCATGCC ATCGACCAGC ⁇ rCAAGGTCA GGTTCTCGAT G3TGCCGGCG ATCCGGTCCT 4140 TGAATACCQG TTCGCCGTCC QGATCCAACT CATCGTAGAA AAAQC ⁇ CGTG CXTTTCGAQCC 4200
  • GCGGCCAT3T AGCTCCAGTT GTAAQGCAGG TAAAACAQCA AAATGAAGAT CGGGATGAGC 5280
  • ATGTCAGT C AACAT GCTG CTGATCGAGA GCTT-TCGAA GATGCGGTAA 033TAQGTAT 7500
  • GGCGTCATCC TGGCCGAACC OGAGGGTCCC
  • GACCAGACGC C03CCTGATC TPCGAATGCC 8880
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • TGCATCGCTT TTCCGGCACT CTAGAGTCTC TAACAGCACA TIGATCTGCC TCTTGCAT3G 360 ATGCACGAAG ACTQGCQGCC TCCCCTCGTC ACAGGCQGCC OGCCTTTGAA ACAAGGAGTG 420
  • GGAACGCACC TTCGACCGGG TAGGCGTATT CQCG3CCACC CACGCTGCCG TGGCQQCCTG 2100
  • CAAGCCCG3C CAGGAGCACG ACCCGAAGGA GTTCACCCAG TGCGTCATTC CCGAGCTGCC 3540
  • CTGCGGAGGA CCTCGCQCGC CGCTCQCGAC CAAGTTCAAG CTCCGCGAAG AACCGTGTCG 3780
  • GGCGCGGATT CACGCCCTCA TTGACGAAOG AGACTTCGCC AAGCCGATCT TCQQCTTCGG 4680
  • GG03GTG03C CAGCTQGAGC CGGTGCCGCG OGGGGCGCGT CAGCCGACGT TGGTCACCGC 5280
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pseudomonas aureofaciens
  • OCAGCCGCTA CAAAAGCCTG TGCGACCCGC GCCTGAAOCC CTGGCAAQCC ATTACT3CGG 1260

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Abstract

L'invention concerne des souches de Pseudomonas génétiquement modifiées pour leur conférer des propriétés de lutte biologique améliorées. Les souches de l'invention sont particulièrement efficaces contre les champignons pathogènes des plantes (espèces Rhizoctonia et Pythium, par exemple), du fait que les souches produisent des quantités améliorées de métabolites antifongiques, tels que la pyrrolnitrine, qui sont actifs contre lesdits pathogènes fongiques. Tant les souches de lutte biologique génétiquement modifiées que les métabolites antifongiques peuvent être utilisés comme matières actives dans des compositions de lutte biologique.
EP97954359A 1996-12-06 1997-12-05 SOUCHES DE $i(PSEUDOMONAS) GENETIQUEMENT MODIFIEES AYANT UNE ACTIVITE DE LUTTE BIOLOGIQUE AMELIOREE Withdrawn EP0941350A1 (fr)

Applications Claiming Priority (5)

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US761258 1996-12-06
US08/761,258 US5756087A (en) 1996-12-06 1996-12-06 Genetically modified Pseudomonas strains with enhanced biocontrol activity
US5830497P 1997-09-09 1997-09-09
US58304P 1997-09-09
PCT/EP1997/006815 WO1998024919A1 (fr) 1996-12-06 1997-12-05 Souches de pseudomonas genetiquement modifiees ayant une activite de lutte biologique amelioree

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US9580719B2 (en) 2007-04-27 2017-02-28 Pfenex, Inc. Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins
US9394571B2 (en) 2007-04-27 2016-07-19 Pfenex Inc. Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins
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US5639949A (en) * 1990-08-20 1997-06-17 Ciba-Geigy Corporation Genes for the synthesis of antipathogenic substances
EP0472494A3 (en) * 1990-08-20 1992-10-21 Ciba Geigy Ag Anti-pathogenic biocontrol agents, genes encoding antibiotics synthesis and the use of said antibiotics
JPH07509605A (ja) * 1992-07-02 1995-10-26 ノバルティス　アクチェンゲゼルシャフト 遺伝子活性化要素

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* Cited by examiner, † Cited by third party
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See references of WO9824919A1 *

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