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James M Ligon - One of the best experts on this subject based on the ideXlab platform.
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Natural products with antifungal activity from Pseudomonas biocontrol bacteria
Pest Management Science, 2000Co-Authors: James M Ligon, Philip E Hammer, Dwight S. Hill, N. R. Torkewitz, Dirk Hofmann, Hans‐joachim Kempf, Karl-heinz Van PéeAbstract:It is now well established that some bacteria and fungi are aggressive colonizers of soil and the roots of plants, and are able to protect plants from infection by soil-borne fungal pathogens. In most cases that have been well studied, the biological mechanisms underlying this phenomenon, known as biocontrol, include the production of antifungal compounds, involving both metabolites and enzymes. Bacteria of the genus Pseudomonas comprise a large group of the active biocontrol strains as a result of their general ability to produce a diverse array of potent antifungal metabolites. These include simple metabolites such as 2,4-diacetylphloroglucinol, phenazine-1-carboxylic acid and Pyrrolnitrin [3-chloro-4-(2'- nitro-3'-chlorophenyl)-pyrrole], as well as the complex macrocyclic lactone, 2,3-de-epoxy-2,3-didehydro-rhizoxin. Study of the biochemistry and mechanism of formation of these metabolites has proved useful in several ways. Pyrrolnitrin is active against Rhizoctonia spp, Fusarium spp, and other plant pathogenic fungi, and it has been used as a lead structure in the development of a new phenylpyrrole agricultural fungicide. In addition, Pyrrolnitrin has been used for years as a model for the study of the mechanisms involved in the chlorination of organic molecules. We have cloned a four-gene cluster from a P fluorescens biocontrol strain that encodes the enzymes required for the production of Pyrrolnitrin. Using these genes and strains mutated in the individual genes, we have elucidated the biochemical pathway by which Pyrrolnitrin is synthesized. Studies of the genes involved in Pyrrolnitrin biosynthesis have demonstrated that a new class of halogenase enzyme is involved in the chlorination reactions in Pyrrolnitrin biosynthesis. The P fluorescens mutants that do not produce Pyrrolnitrin have been used to demonstrate clearly the important role of Pyrrolnitrin in the overall biocontrol activity shown by the strain. In addition, we have modified the Pyrrolnitrin genes within the P fluorescens strain, which has resulted in significant increases in the production of this metabolite. The strains which overproduce Pyrrolnitrin are also significantly more active than the wild-type strain in biocontrol. These studies suggest that the biocontrol activity of bacteria can be dramatically increased to rival the activity of commercial chemical fungicides through directed genetic modification. Bacteria remain important sources of natural products with diverse activities. In order to capture the full potential of these compounds, it will be necessary in the future to take a multidisciplinary approach to their study and development.
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Conservation of the Pyrrolnitrin biosynthetic gene cluster among six Pyrrolnitrin-producing strains
FEMS microbiology letters, 1999Co-Authors: Philip E Hammer, James M Ligon, Wassily Burd, D. Steven Hill, Karl-heinz Van PéeAbstract:The prnABCD gene cluster from Pseudomonas fluorescens encodes the biosynthetic pathway for Pyrrolnitrin, a secondary metabolite derived from tryptophan which has strong anti-fungal activity. We used the prn genes from P. fluorescens strain BL915 as a probe to clone and sequence homologous genes from three other Pseudomonas strains, Burkholderia cepacia and Myxococcus fulvus. With the exception of the prnA gene from M. fulvus, the deduced amino acid sequences were >59% similar among the strains, indicating that the biochemical pathway for Pyrrolnitrin biosynthesis is highly conserved. The prnA gene from M. fulvus is about 45% similar to prnA from the other strains and contains regions which are highly conserved among all six strains.
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functions encoded by Pyrrolnitrin biosynthetic genes from pseudomonas fluorescens
Journal of Bacteriology, 1998Co-Authors: Sabine Kirner, Philip E Hammer, Steven D Hill, Annett Altmann, Ilona Fischer, Laura J Weislo, Mike Lanahan, James M LigonAbstract:Pyrrolnitrin is a secondary metabolite derived from tryptophan and has strong antifungal activity. Recently we described four genes, prnABCD, from Pseudomonas fluorescens that encode the biosynthesis of Pyrrolnitrin. In the work presented here, we describe the function of each prn gene product. The four genes encode proteins identical in size and serology to proteins present in wild-type Pseudomonas fluorescens, but absent from a mutant from which the entire prn gene region had been deleted. The prnA gene product catalyzes the chlorination of l-tryptophan to form 7-chloro-l-tryptophan. The prnB gene product catalyzes a ring rearrangement and decarboxylation to convert 7-chloro-l-tryptophan to monodechloroaminoPyrrolnitrin. The prnC gene product chlorinates monodechloroaminoPyrrolnitrin at the 3 position to form aminoPyrrolnitrin. The prnD gene product catalyzes the oxidation of the amino group of aminoPyrrolnitrin to a nitro group to form Pyrrolnitrin. The organization of the prn genes in the operon is identical to the order of the reactions in the biosynthetic pathway.
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four genes from pseudomonas fluorescens that encode the biosynthesis of Pyrrolnitrin
Applied and Environmental Microbiology, 1997Co-Authors: Philip E Hammer, Dwight Steven Hill, James M LigonAbstract:Pyrrolnitrin is a secondary metabolite of Pseudomonas and Burkholderia sp. strains with strong antifungal activity. Production of Pyrrolnitrin has been correlated with the ability of some bacteria to control plant diseases caused by fungal pathogens, including the damping-off pathogen Rhizoctonia solani. Pseudomonas fluorescens BL915 has been reported to produce Pyrrolnitrin and to be an effective biocontrol agent for this pathogen. We have isolated a 32-kb genomic DNA fragment from this strain that contains genes involved in the biosynthesis of Pyrrolnitrin. Marker-exchange mutagenesis of this DNA with Tn5 revealed the presence of a 6.2-kb region that contains genes required for the synthesis of Pyrrolnitrin. The nucleotide sequence of the 6.2-kb region was determined and found to contain a cluster of four genes that are required for the production of Pyrrolnitrin. Deletion mutations in any of the four genes resulted in a Pyrrolnitrin-nonproducing phenotype. The putative coding sequences of the four individual genes were cloned by PCR and fused to the tac promoter from Escherichia coli. In each case, the appropriate tac promoter-Pyrrolnitrin gene fusion was shown to complement the Pyrrolnitrin-negative phenotype of the corresponding deletion mutant. Transfer of the four gene cluster to E. coli resulted in the production of Pyrrolnitrin by this organism, thereby demonstrating that the four genes are sufficient for the production of this metabolite and represent all of the genes required to encode the pathway for Pyrrolnitrin biosynthesis.
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Cloning ofGenesInvolved intheSynthesis ofPyrrolnitrin fromPseudomonas fluorescens andRoleofPyrrolnitrin Synthesis inBiological Control ofPlant Disease
1994Co-Authors: Dwight S. Hill, J. I. Stein, N. R. Torkewitz, A. M. Morse, C. R. Howell, J. P. Pachlatko, James M LigonAbstract:A soil isolate ofPseudomonas fluorescens (BL915) was showntobean effective antagonist ofRhizoctonia solani-induced damping-off ofcotton. Investigation ofthebiological basis ofthisantagonism revealed thatthe strain produces Pyrrolnitrin, a secondary metabolite knowntoinhibit R.solani andotherfungi. Mutantsof strain BL915thatdidnotproduce Pyrrolnitrin anddidnotsuppressdamping-off ofcotton byR.solani were generated byexposuretoN-methyl-N'-nitro-N-nitrosoguanidine. A generegion that was capable ofrestoring Pyrrolnitrin production tothenon-Pyrrolnitrin-producing mutantsandofconferring thisability upon two other P.fluorescens strains nototherwise knowntoproduce this compound ortobecapable ofsuppressing damping-off causedbyR.solani was isolated fromstrain BL915.Thenon-Pyrrolnitrin-producing strains (mutants ofBL915andtheothertwoP.fluorescens strains) whichsynthesized Pyrrolnitrin after the
Karl-heinz Van Pée - One of the best experts on this subject based on the ideXlab platform.
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Preliminary crystallographic characterization of PrnB, the second enzyme in the Pyrrolnitrin biosynthetic pathway.
Acta crystallographica. Section F Structural biology and crystallization communications, 2006Co-Authors: Walter De Laurentis, Karl-heinz Van Pée, Khim Leang, Katrin Hahn, Bianca Podemski, Ariane Adam, Sonja Kroschwald, Lester G. Carter, James H. NaismithAbstract:Pyrrolnitrin is the active ingredient of drugs for the treatment of superficial fungal infections and was used as a lead structure for the development of fludioxonil. It is an effective agent for plant diseases caused by the fungal pathogen Rhizoctonia solani. Pyrrolnitrin is made in four steps, the second of which, catalyzed by PrnB, is a novel chemical rearrangement of 7-chlorotryptophan. PrnB was overproduced in Pseudomonas fluorescens (BL915) and well diffracting crystals were obtained of a triple cysteine-to-serine mutant by sitting-drop vapour diffusion. Crystals grown in the presence of L-7-chlorotryptophan, D-tryptophan and L-tryptophan are reported. Data sets for each are reported with high-resolution limits of 2.0, 1.75 and 1.75 A, respectively. Two crystals (PrnB in the presence of D-tryptophan and L-7-chlorotryptophan) belong to space group C2 with similar unit-cell parameters (a = 68.6, b = 79.5, c = 92.7 A, alpha = gamma = 90.0, beta = 103.8 degrees). Crystals grown in the presence of L-tryptophan belong to space group C222(1) and have unit-cell parameters a = 67.7, b = 80.1, c = 129.5 A. All crystals contain a monomer in the asymmetric unit.
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Preliminary crystallographic characterization of PrnB, the second enzyme in the Pyrrolnitrin biosynthetic pathway
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2006Co-Authors: Walter De Laurentis, Karl-heinz Van Pée, Khim Leang, Katrin Hahn, Bianca Podemski, Ariane Adam, Sonja Kroschwald, Lester G. Carter, James H. NaismithAbstract:Pyrrolnitrin is the active ingredient of drugs for the treatment of superficial fungal infections and was used as a lead structure for the development of fludioxonil. It is an effective agent for plant diseases caused by the fungal pathogen Rhizoctonia solani. Pyrrolnitrin is made in four steps, the second of which, catalyzed by PrnB, is a novel chemical rearrangement of 7-chlorotryptophan. PrnB was overproduced in Pseudomonas fluorescens (BL915) and well diffracting crystals were obtained of a triple cysteine-to-serine mutant by sitting-drop vapour diffusion. Crystals grown in the presence of l-7-chlorotryptophan, d-tryptophan and l-tryptophan are reported. Data sets for each are reported with high-resolution limits of 2.0, 1.75 and 1.75 A, respectively. Two crystals (PrnB in the presence of d-tryptophan and l-7-chlorotryptophan) belong to space group C2 with similar unit-cell parameters (a = 68.6, b = 79.5, c = 92.7 A, α = γ = 90.0, β = 103.8°). Crystals grown in the presence of l-tryptophan belong to space group C2221 and have unit-cell parameters a = 67.7, b = 80.1, c = 129.5 A. All crystals contain a monomer in the asymmetric unit.
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A flavin-dependent tryptophan 6-halogenase and its use in modification of Pyrrolnitrin biosynthesis
Biocatalysis and Biotransformation, 2006Co-Authors: Corina Seibold, Helge Schnerr, Julia Rumpf, Andrea Kunzendorf, Catharina Hatscher, Tobias Wage, Aliz J. Ernyei, Changjiang Dong, James H. Naismith, Karl-heinz Van PéeAbstract:Regioselective halogenation of electron rich substrates is catalysed by flavin-dependent halogenases. Thienodolin produced by Streptomyces albogriseolus contains a chlorine atom in the 6-position of the indole ring system and is believed to be derived from tryptophan. Using the gene of the tryptophan 7-halogenase (PrnA) from the Pyrrolnitrin biosynthetic gene cluster the gene for a tryptophan 6-halogenase was cloned, sequenced and heterologously overexpressed in Pseudomonas strains. In vitro activity of the purified enzyme could only be shown in a two-component enzyme system consisting of the halogenase, a flavin reductase, NADH, FAD and halide ions. The enzyme catalyses the regioselective chlorination and bromination of l- and d-tryptophan. In its native form the enzyme is probably a homodimer with a relative molecular mass of the subunits of 63 600 (including the His-tag). Transformation of the Pyrrolnitrin producer Pseudomonas chlororaphis ACN with a plasmid containing the tryptophan 6-halogenase gene ...
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Natural products with antifungal activity from Pseudomonas biocontrol bacteria
Pest Management Science, 2000Co-Authors: James M Ligon, Philip E Hammer, Dwight S. Hill, N. R. Torkewitz, Dirk Hofmann, Hans‐joachim Kempf, Karl-heinz Van PéeAbstract:It is now well established that some bacteria and fungi are aggressive colonizers of soil and the roots of plants, and are able to protect plants from infection by soil-borne fungal pathogens. In most cases that have been well studied, the biological mechanisms underlying this phenomenon, known as biocontrol, include the production of antifungal compounds, involving both metabolites and enzymes. Bacteria of the genus Pseudomonas comprise a large group of the active biocontrol strains as a result of their general ability to produce a diverse array of potent antifungal metabolites. These include simple metabolites such as 2,4-diacetylphloroglucinol, phenazine-1-carboxylic acid and Pyrrolnitrin [3-chloro-4-(2'- nitro-3'-chlorophenyl)-pyrrole], as well as the complex macrocyclic lactone, 2,3-de-epoxy-2,3-didehydro-rhizoxin. Study of the biochemistry and mechanism of formation of these metabolites has proved useful in several ways. Pyrrolnitrin is active against Rhizoctonia spp, Fusarium spp, and other plant pathogenic fungi, and it has been used as a lead structure in the development of a new phenylpyrrole agricultural fungicide. In addition, Pyrrolnitrin has been used for years as a model for the study of the mechanisms involved in the chlorination of organic molecules. We have cloned a four-gene cluster from a P fluorescens biocontrol strain that encodes the enzymes required for the production of Pyrrolnitrin. Using these genes and strains mutated in the individual genes, we have elucidated the biochemical pathway by which Pyrrolnitrin is synthesized. Studies of the genes involved in Pyrrolnitrin biosynthesis have demonstrated that a new class of halogenase enzyme is involved in the chlorination reactions in Pyrrolnitrin biosynthesis. The P fluorescens mutants that do not produce Pyrrolnitrin have been used to demonstrate clearly the important role of Pyrrolnitrin in the overall biocontrol activity shown by the strain. In addition, we have modified the Pyrrolnitrin genes within the P fluorescens strain, which has resulted in significant increases in the production of this metabolite. The strains which overproduce Pyrrolnitrin are also significantly more active than the wild-type strain in biocontrol. These studies suggest that the biocontrol activity of bacteria can be dramatically increased to rival the activity of commercial chemical fungicides through directed genetic modification. Bacteria remain important sources of natural products with diverse activities. In order to capture the full potential of these compounds, it will be necessary in the future to take a multidisciplinary approach to their study and development.
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Conservation of the Pyrrolnitrin biosynthetic gene cluster among six Pyrrolnitrin-producing strains
FEMS microbiology letters, 1999Co-Authors: Philip E Hammer, James M Ligon, Wassily Burd, D. Steven Hill, Karl-heinz Van PéeAbstract:The prnABCD gene cluster from Pseudomonas fluorescens encodes the biosynthetic pathway for Pyrrolnitrin, a secondary metabolite derived from tryptophan which has strong anti-fungal activity. We used the prn genes from P. fluorescens strain BL915 as a probe to clone and sequence homologous genes from three other Pseudomonas strains, Burkholderia cepacia and Myxococcus fulvus. With the exception of the prnA gene from M. fulvus, the deduced amino acid sequences were >59% similar among the strains, indicating that the biochemical pathway for Pyrrolnitrin biosynthesis is highly conserved. The prnA gene from M. fulvus is about 45% similar to prnA from the other strains and contains regions which are highly conserved among all six strains.
Wojciech J. Janisiewicz - One of the best experts on this subject based on the ideXlab platform.
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Postharvest control of Botrytis cinerea on cut rose flowers with Pyrrolnitrin
Plant Disease, 1993Co-Authors: P. E. Hammer, Kathleen B. Evensen, Wojciech J. JanisiewiczAbstract:Pyrrolnitrin, an antibiotic isolated from Pseudomonas cepacia, was tested for postharvest control of Botrytis cinerea infections on cut Sonia and Royalty rose flowers. After Pyrrolnitrin was applied as a bud dip, buds were inoculated with conidia of B. cinerea and stored for 7 days at 2 C. Dip treatments of 12-200 mg/L significantly reduced lesion development during storage at 2 C and promoted poststorage fresh weight gain (an index of cut flower quality). No phytotoxicity was observed on leaves or petals at concentrations of Pyrrolnitrin up to 200 mg/L. Dip treatment with 100 mg/L reduced lesion development by about 90% compared to inoculated control flowers and prevented poststorage flower rot (.)
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Pyrrolnitrin captan benomyl and high co2 enhance raspberry shelf life at 0 or 18c
Journal of the American Society for Horticultural Science, 1992Co-Authors: Barbara L. Goulart, Philip E Hammer, Kathleen B. Evensen, Wojciech J. Janisiewicz, Fumiomi TakedaAbstract:The effects of preharvest applications of Pyrrolnitrin (a biologically derived fungicide) on postharvest longevity of ‘Bristol’ black raspberry (Rubus occidentals L.) and ‘Heritage’ red raspberry [R. idaeus L. var. strigosus (Michx.) Maxim] were evaluated at two storage temperatures. Preharvest fungicide treatments were 200 mg Pyrrolnitrin/liter, a standard fungicide treatment (captan + benomyl or iprodione) or a distilled water control applied 1 day before first harvest. Black raspberries were stored at 18 or 0 ± lC in air or 20% CO2. Red raspberries were stored at the same temperatures in air only. Pyrrolnitrin-treated berries often had less gray mold (Botrytis cinerea Pers. ex Fr.) in storage than the control but more than berries treated with the standard fungicides. Storage in a modified atmosphere of 20% CO2 greatly improved postharvest quality of black raspberries at both storage temperatures by reducing gray mold development. The combination of standard fungicide or Pyrrolnitrin, high CO2, and low temperature resulted in more than 2 weeks of storage with less than 5% disease on black raspberries; however, discoloration limited marketability after 8 days under these conditions. Chemical names used: 3-chloro-4-(2’-nitro3’-chlorophenyl) -pyrrole (Pyrrolnitrin); N-trichloromethylthio-4-cyclohexene-l12-dicarboximide (captan); methyl 1(butylcarbamoyl) -2-benzimidazolecarbamate) (benomyl); 3-(3,5 -dichlorophenyl) -N-(l-methylethyl -2,4-dioxo-l-imidazolidinecarboxamide (Rovral, iprodione). Raspberry production for fresh-market consumption is severely limited by the rapid deterioration of the fruit. While the raspberry fruit has morphological and physiological characteristics that account for a portion of its short postharvest life, specifically loosely connected drupelets, an open cavity that predisposes it to crushing, and a high postharvest rate of respiration; the most common cause of postharvest decline is gray mold fruit rot (Janisiewicz, 1988). While the benefits of modified atmosphere storage are well documented on strawberries (Couey and Wells, 1970; Harvey, 1982a, 1982b), research is sparse and less conclusive on raspberries. Winter et al. (1939) reported increased shelf life of raspberries at 10 to 15C using an initial CO2 concentration of 30%. Smith (1958) found that modified atmospheres with 20% to 25% CO2 slowed ripening and reduced decay. Preharvest applications of fungicides have been shown to control postharvest gray mold occurrence (Freeman and Pepin, 1976). However, the development of fungicide-resistant fungal populations and increasingly stringent governmental regulation limit the use of currently available fungicides. Biological control agents and naturally produced compounds may offer alternative means of controlling fungal infections in raspberries. While little biological control research has been conducted on raspberries, Tronsmo and Dennis (1977) worked with strawberry and found that several microbial organisms, including Trichodenna spp., Gliocladium virens, and Hypocrea semiorbitis, reduced gray mold spoilage in fruit in both the field and storage when applied from early flowering until 2 weeks before harvest. However, postharvest incidence of fruit rot caused by Mucor spp. increased (Janisiewicz and Roitman, 1988). Pyrrolnitrin, isolated from Pseudomonas cepacia, a gramfor publication 7 June 1991. Accepted for publication 27 Sept. 1991. Tire ublishing this paper was defrayed in part by the payment of page Under postal regulations, this paper therefore must be hereby marked ment solely to indicate this fact. r. Soc. Hort. Sci. 117(2):265-270. 1992. negative bacterium, has broad-spectrum antibiotic properties (Imanaka et al., 1965a, 1965b; Janisiewicz and Roitman, 1988). It inhibits growth of fungi, yeasts, and gram-positive bacteria and has low mammalian toxicity (Arima et al., 1965). Takeda et al. (1990) reported inhibition of fungal growth on strawberries treated with a postharvest dip of 250 ppm Pyrrolnitrin and stored at 18C, or at 0C followed by 18C. Unlike some other biocontrol agents, Pyrrolnitrin did not increase the incidence of fruit decay caused by Mucor. The objective of this research was to evaluate the effect of preharvest applications of Pyrrolnitrin on the postharvest longevity of raspberries under various postharvest conditions. Materials and Methods Experiments were conducted in 1989 and 1990 at the Russell E. Larson Research Center in Rock Springs, Pa. For Expts. 1 and 2 (1989), a mature ‘Bristol’ black raspberry planting grown in a hedgerow system according to commercial recommendations was sprayed with recommended rates of captan + benomyl weekly for fungal control until 2 weeks before harvest. At this time, field treatments consisting of 200 ppm Pyrrolnitrin (pure crystals were dissolved in 5 ml of methanol, and distilled water was added for a total volume of 1 liter) plus 0.5 ml Tween 20 wetting agent; captan + benomyl at 1.8 + 0.45 g a.i./liter; or distilled water (control) were applied until drip. Two spray applications were made, on 6 and 13 July 1989. In 1990, ‘Heritage’ red raspberries were harvested from a 3-year-old planting. Fungicide treatments were applied on 13 Sept. For all experiments, berries were harvested 1, 4, and 6 days after treatment application, cooled to 5C immediately after harvest in a forcedair cooler, and kept cold during transport and handling. Each postharvest experimental unit consisted of a 235 x 150 mm plastic tray (a candy box insert) with 24 separate compartments. Individual berries were placed in tray compartments, so that each berry was clearly visible and did not touch the surrounding berries.
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Pyrrolnitrin, CAPTAN + BENOMYL, AND HIGH CO2 ENHANCE RASPBERRY SHELF LIFE AT 0 OR 18C
Journal of the American Society for Horticultural Science, 1992Co-Authors: Barbara L. Goulart, Philip E Hammer, Kathleen B. Evensen, Wojciech J. Janisiewicz, Fumiomi TakedaAbstract:The effects of preharvest applications of Pyrrolnitrin (a biologically derived fungicide) on postharvest longevity of ‘Bristol’ black raspberry (Rubus occidentals L.) and ‘Heritage’ red raspberry [R. idaeus L. var. strigosus (Michx.) Maxim] were evaluated at two storage temperatures. Preharvest fungicide treatments were 200 mg Pyrrolnitrin/liter, a standard fungicide treatment (captan + benomyl or iprodione) or a distilled water control applied 1 day before first harvest. Black raspberries were stored at 18 or 0 ± lC in air or 20% CO2. Red raspberries were stored at the same temperatures in air only. Pyrrolnitrin-treated berries often had less gray mold (Botrytis cinerea Pers. ex Fr.) in storage than the control but more than berries treated with the standard fungicides. Storage in a modified atmosphere of 20% CO2 greatly improved postharvest quality of black raspberries at both storage temperatures by reducing gray mold development. The combination of standard fungicide or Pyrrolnitrin, high CO2, and low temperature resulted in more than 2 weeks of storage with less than 5% disease on black raspberries; however, discoloration limited marketability after 8 days under these conditions. Chemical names used: 3-chloro-4-(2’-nitro3’-chlorophenyl) -pyrrole (Pyrrolnitrin); N-trichloromethylthio-4-cyclohexene-l12-dicarboximide (captan); methyl 1(butylcarbamoyl) -2-benzimidazolecarbamate) (benomyl); 3-(3,5 -dichlorophenyl) -N-(l-methylethyl -2,4-dioxo-l-imidazolidinecarboxamide (Rovral, iprodione). Raspberry production for fresh-market consumption is severely limited by the rapid deterioration of the fruit. While the raspberry fruit has morphological and physiological characteristics that account for a portion of its short postharvest life, specifically loosely connected drupelets, an open cavity that predisposes it to crushing, and a high postharvest rate of respiration; the most common cause of postharvest decline is gray mold fruit rot (Janisiewicz, 1988). While the benefits of modified atmosphere storage are well documented on strawberries (Couey and Wells, 1970; Harvey, 1982a, 1982b), research is sparse and less conclusive on raspberries. Winter et al. (1939) reported increased shelf life of raspberries at 10 to 15C using an initial CO2 concentration of 30%. Smith (1958) found that modified atmospheres with 20% to 25% CO2 slowed ripening and reduced decay. Preharvest applications of fungicides have been shown to control postharvest gray mold occurrence (Freeman and Pepin, 1976). However, the development of fungicide-resistant fungal populations and increasingly stringent governmental regulation limit the use of currently available fungicides. Biological control agents and naturally produced compounds may offer alternative means of controlling fungal infections in raspberries. While little biological control research has been conducted on raspberries, Tronsmo and Dennis (1977) worked with strawberry and found that several microbial organisms, including Trichodenna spp., Gliocladium virens, and Hypocrea semiorbitis, reduced gray mold spoilage in fruit in both the field and storage when applied from early flowering until 2 weeks before harvest. However, postharvest incidence of fruit rot caused by Mucor spp. increased (Janisiewicz and Roitman, 1988). Pyrrolnitrin, isolated from Pseudomonas cepacia, a gramfor publication 7 June 1991. Accepted for publication 27 Sept. 1991. Tire ublishing this paper was defrayed in part by the payment of page Under postal regulations, this paper therefore must be hereby marked ment solely to indicate this fact. r. Soc. Hort. Sci. 117(2):265-270. 1992. negative bacterium, has broad-spectrum antibiotic properties (Imanaka et al., 1965a, 1965b; Janisiewicz and Roitman, 1988). It inhibits growth of fungi, yeasts, and gram-positive bacteria and has low mammalian toxicity (Arima et al., 1965). Takeda et al. (1990) reported inhibition of fungal growth on strawberries treated with a postharvest dip of 250 ppm Pyrrolnitrin and stored at 18C, or at 0C followed by 18C. Unlike some other biocontrol agents, Pyrrolnitrin did not increase the incidence of fruit decay caused by Mucor. The objective of this research was to evaluate the effect of preharvest applications of Pyrrolnitrin on the postharvest longevity of raspberries under various postharvest conditions. Materials and Methods Experiments were conducted in 1989 and 1990 at the Russell E. Larson Research Center in Rock Springs, Pa. For Expts. 1 and 2 (1989), a mature ‘Bristol’ black raspberry planting grown in a hedgerow system according to commercial recommendations was sprayed with recommended rates of captan + benomyl weekly for fungal control until 2 weeks before harvest. At this time, field treatments consisting of 200 ppm Pyrrolnitrin (pure crystals were dissolved in 5 ml of methanol, and distilled water was added for a total volume of 1 liter) plus 0.5 ml Tween 20 wetting agent; captan + benomyl at 1.8 + 0.45 g a.i./liter; or distilled water (control) were applied until drip. Two spray applications were made, on 6 and 13 July 1989. In 1990, ‘Heritage’ red raspberries were harvested from a 3-year-old planting. Fungicide treatments were applied on 13 Sept. For all experiments, berries were harvested 1, 4, and 6 days after treatment application, cooled to 5C immediately after harvest in a forcedair cooler, and kept cold during transport and handling. Each postharvest experimental unit consisted of a 235 x 150 mm plastic tray (a candy box insert) with 24 separate compartments. Individual berries were placed in tray compartments, so that each berry was clearly visible and did not touch the surrounding berries.
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Postharvest control of blue mold and gray mold of apples and pears by dip treatment with Pyrrolnitrin, a metabolite of Pseudomonas cepacia
Plant Disease, 1991Co-Authors: Wojciech J. Janisiewicz, J. Roitman, L. Yourman, Noreen MahoneyAbstract:The antifungal compound Pyrrolnitrin, isolated from Pseudomonas cepacia, an antagonist known to control gray mold (incited by Botrytis cinerea) and blue mold (incited by Penicillium expansum) of apples and pears, was assayed for its efficacy in controlling these diseases on wounded fruit at two temperatures (2 and 24 C). The compound was applied to wounded fruit after harvest at concentrations ranging from 6 to 200 μg/ml in dip solutions containing conidia of P. expansum or B. cinerea (1 × 10 4 conidia per milliliter). Pyrrolnitrin provided effective control of both diseases on apples and pears (.)
Xiaoguang Liu - One of the best experts on this subject based on the ideXlab platform.
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role of the rna binding protein hfq in serratia plymuthica
Frontiers in Bioscience, 2012Co-Authors: Min Zhou, Kexiang Gao, Jun Zeng, Yunfei Duan, Xiaoguang LiuAbstract:The RNA-binding protein Hfq has been well studied as a global post-transcriptional regulator which controls diverse cellular processes in bacteria. However, the function in the genus of Serratia has remained unexplored. Here we show that beyond mutation in Hfq resulting in their growth defects, Hfq has global effects on a variety of biocontrol-related phenotypes in the endophytic strain G3 of Serratia plymuthica, including antifungal activity, production of exoenzymes, as well as motility and biofilm formation. Especially for the first time, Hfq is observed to control biosynthesis of auxin indole-3-acetic acid (IAA) and antibiotic Pyrrolnitrin (PRN), which are key determinants responsible for plant growth promotion and suppression of phytopathogens, respectively by G3. Additionally, Hfq is also required for the production of RpoS, a major stress sigma factor in G3. In contrast to E. coli, translation of hfq in G3 is positively autoregulated. Further investigation of the molecular mechanisms involved in regulation of IAA and Pyrrolnitrin production by Hfq and its role in the regulatory networks of G3 will help to optimize the beneficial plant-microbe interactions.
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Quorum-sensing signaling is required for production of the antibiotic Pyrrolnitrin in a rhizospheric biocontrol strain of Serratia plymuthica.
FEMS microbiology letters, 2007Co-Authors: Xiaoguang Liu, Mohammed Bimerew, Henry Müller, Marianna Ovadis, Leo Eberl, Gabriele Berg, Leonid CherninAbstract:One mechanism that bacteria have adopted to regulate the production of antimicrobial compounds is population-density-dependent LuxRI-type quorum sensing (QS), exploiting the production of N-acyl homoserine lactone (AHL) autoinducer signals. In biocontrol bacteria, most known cases involve the AHL control of phenazine antibiotics production by rhizospheric pseudomonads. This work is the first to demonstrate that phenazines are not the only group of biocontrol-related antibiotics whose production is regulated by QS systems. Strain HRO-C48 of Serratia plymuthica isolated from the rhizosphere of oilseed rape and described as a chitinolytic bacterium, which protects crops against Verticillium wilt, was also shown to produce wide-range antibiotic Pyrrolnitrin and several AHLs, including N-butanoyl-HSL, N-hexanoyl-HSL and N-3-oxo-hexanoyl-HSL (OHHL). The genes splI and splR, which are analogues of luxI and luxR genes from other Gram-negative bacteria, were cloned and sequenced. The mutant AHL-4 (splI::miniTn5) was simultaneously deficient in the production of AHLs and Pyrrolnitrin, as well as in its ability to suppress the growth of several fungal plant pathogens in vitro. However, Pyrrolnitrin production could be restored in this mutant by introduction of the splIR genes cloned into a plasmid or by addition of the conditioned medium from strain C48 or OHHL standard to the growth medium.
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Natural products with antifungal activity from Pseudomonas biocontrol bacteria
Pest Management Science, 2000Co-Authors: James M Ligon, Philip E Hammer, Dwight S. Hill, N. R. Torkewitz, Dirk Hofmann, Hans‐joachim Kempf, Karl-heinz Van PéeAbstract:It is now well established that some bacteria and fungi are aggressive colonizers of soil and the roots of plants, and are able to protect plants from infection by soil-borne fungal pathogens. In most cases that have been well studied, the biological mechanisms underlying this phenomenon, known as biocontrol, include the production of antifungal compounds, involving both metabolites and enzymes. Bacteria of the genus Pseudomonas comprise a large group of the active biocontrol strains as a result of their general ability to produce a diverse array of potent antifungal metabolites. These include simple metabolites such as 2,4-diacetylphloroglucinol, phenazine-1-carboxylic acid and Pyrrolnitrin [3-chloro-4-(2'- nitro-3'-chlorophenyl)-pyrrole], as well as the complex macrocyclic lactone, 2,3-de-epoxy-2,3-didehydro-rhizoxin. Study of the biochemistry and mechanism of formation of these metabolites has proved useful in several ways. Pyrrolnitrin is active against Rhizoctonia spp, Fusarium spp, and other plant pathogenic fungi, and it has been used as a lead structure in the development of a new phenylpyrrole agricultural fungicide. In addition, Pyrrolnitrin has been used for years as a model for the study of the mechanisms involved in the chlorination of organic molecules. We have cloned a four-gene cluster from a P fluorescens biocontrol strain that encodes the enzymes required for the production of Pyrrolnitrin. Using these genes and strains mutated in the individual genes, we have elucidated the biochemical pathway by which Pyrrolnitrin is synthesized. Studies of the genes involved in Pyrrolnitrin biosynthesis have demonstrated that a new class of halogenase enzyme is involved in the chlorination reactions in Pyrrolnitrin biosynthesis. The P fluorescens mutants that do not produce Pyrrolnitrin have been used to demonstrate clearly the important role of Pyrrolnitrin in the overall biocontrol activity shown by the strain. In addition, we have modified the Pyrrolnitrin genes within the P fluorescens strain, which has resulted in significant increases in the production of this metabolite. The strains which overproduce Pyrrolnitrin are also significantly more active than the wild-type strain in biocontrol. These studies suggest that the biocontrol activity of bacteria can be dramatically increased to rival the activity of commercial chemical fungicides through directed genetic modification. Bacteria remain important sources of natural products with diverse activities. In order to capture the full potential of these compounds, it will be necessary in the future to take a multidisciplinary approach to their study and development.
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Conservation of the Pyrrolnitrin biosynthetic gene cluster among six Pyrrolnitrin-producing strains
FEMS microbiology letters, 1999Co-Authors: Philip E Hammer, James M Ligon, Wassily Burd, D. Steven Hill, Karl-heinz Van PéeAbstract:The prnABCD gene cluster from Pseudomonas fluorescens encodes the biosynthetic pathway for Pyrrolnitrin, a secondary metabolite derived from tryptophan which has strong anti-fungal activity. We used the prn genes from P. fluorescens strain BL915 as a probe to clone and sequence homologous genes from three other Pseudomonas strains, Burkholderia cepacia and Myxococcus fulvus. With the exception of the prnA gene from M. fulvus, the deduced amino acid sequences were >59% similar among the strains, indicating that the biochemical pathway for Pyrrolnitrin biosynthesis is highly conserved. The prnA gene from M. fulvus is about 45% similar to prnA from the other strains and contains regions which are highly conserved among all six strains.
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functions encoded by Pyrrolnitrin biosynthetic genes from pseudomonas fluorescens
Journal of Bacteriology, 1998Co-Authors: Sabine Kirner, Philip E Hammer, Steven D Hill, Annett Altmann, Ilona Fischer, Laura J Weislo, Mike Lanahan, James M LigonAbstract:Pyrrolnitrin is a secondary metabolite derived from tryptophan and has strong antifungal activity. Recently we described four genes, prnABCD, from Pseudomonas fluorescens that encode the biosynthesis of Pyrrolnitrin. In the work presented here, we describe the function of each prn gene product. The four genes encode proteins identical in size and serology to proteins present in wild-type Pseudomonas fluorescens, but absent from a mutant from which the entire prn gene region had been deleted. The prnA gene product catalyzes the chlorination of l-tryptophan to form 7-chloro-l-tryptophan. The prnB gene product catalyzes a ring rearrangement and decarboxylation to convert 7-chloro-l-tryptophan to monodechloroaminoPyrrolnitrin. The prnC gene product chlorinates monodechloroaminoPyrrolnitrin at the 3 position to form aminoPyrrolnitrin. The prnD gene product catalyzes the oxidation of the amino group of aminoPyrrolnitrin to a nitro group to form Pyrrolnitrin. The organization of the prn genes in the operon is identical to the order of the reactions in the biosynthetic pathway.
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four genes from pseudomonas fluorescens that encode the biosynthesis of Pyrrolnitrin
Applied and Environmental Microbiology, 1997Co-Authors: Philip E Hammer, Dwight Steven Hill, James M LigonAbstract:Pyrrolnitrin is a secondary metabolite of Pseudomonas and Burkholderia sp. strains with strong antifungal activity. Production of Pyrrolnitrin has been correlated with the ability of some bacteria to control plant diseases caused by fungal pathogens, including the damping-off pathogen Rhizoctonia solani. Pseudomonas fluorescens BL915 has been reported to produce Pyrrolnitrin and to be an effective biocontrol agent for this pathogen. We have isolated a 32-kb genomic DNA fragment from this strain that contains genes involved in the biosynthesis of Pyrrolnitrin. Marker-exchange mutagenesis of this DNA with Tn5 revealed the presence of a 6.2-kb region that contains genes required for the synthesis of Pyrrolnitrin. The nucleotide sequence of the 6.2-kb region was determined and found to contain a cluster of four genes that are required for the production of Pyrrolnitrin. Deletion mutations in any of the four genes resulted in a Pyrrolnitrin-nonproducing phenotype. The putative coding sequences of the four individual genes were cloned by PCR and fused to the tac promoter from Escherichia coli. In each case, the appropriate tac promoter-Pyrrolnitrin gene fusion was shown to complement the Pyrrolnitrin-negative phenotype of the corresponding deletion mutant. Transfer of the four gene cluster to E. coli resulted in the production of Pyrrolnitrin by this organism, thereby demonstrating that the four genes are sufficient for the production of this metabolite and represent all of the genes required to encode the pathway for Pyrrolnitrin biosynthesis.
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Pyrrolnitrin captan benomyl and high co2 enhance raspberry shelf life at 0 or 18c
Journal of the American Society for Horticultural Science, 1992Co-Authors: Barbara L. Goulart, Philip E Hammer, Kathleen B. Evensen, Wojciech J. Janisiewicz, Fumiomi TakedaAbstract:The effects of preharvest applications of Pyrrolnitrin (a biologically derived fungicide) on postharvest longevity of ‘Bristol’ black raspberry (Rubus occidentals L.) and ‘Heritage’ red raspberry [R. idaeus L. var. strigosus (Michx.) Maxim] were evaluated at two storage temperatures. Preharvest fungicide treatments were 200 mg Pyrrolnitrin/liter, a standard fungicide treatment (captan + benomyl or iprodione) or a distilled water control applied 1 day before first harvest. Black raspberries were stored at 18 or 0 ± lC in air or 20% CO2. Red raspberries were stored at the same temperatures in air only. Pyrrolnitrin-treated berries often had less gray mold (Botrytis cinerea Pers. ex Fr.) in storage than the control but more than berries treated with the standard fungicides. Storage in a modified atmosphere of 20% CO2 greatly improved postharvest quality of black raspberries at both storage temperatures by reducing gray mold development. The combination of standard fungicide or Pyrrolnitrin, high CO2, and low temperature resulted in more than 2 weeks of storage with less than 5% disease on black raspberries; however, discoloration limited marketability after 8 days under these conditions. Chemical names used: 3-chloro-4-(2’-nitro3’-chlorophenyl) -pyrrole (Pyrrolnitrin); N-trichloromethylthio-4-cyclohexene-l12-dicarboximide (captan); methyl 1(butylcarbamoyl) -2-benzimidazolecarbamate) (benomyl); 3-(3,5 -dichlorophenyl) -N-(l-methylethyl -2,4-dioxo-l-imidazolidinecarboxamide (Rovral, iprodione). Raspberry production for fresh-market consumption is severely limited by the rapid deterioration of the fruit. While the raspberry fruit has morphological and physiological characteristics that account for a portion of its short postharvest life, specifically loosely connected drupelets, an open cavity that predisposes it to crushing, and a high postharvest rate of respiration; the most common cause of postharvest decline is gray mold fruit rot (Janisiewicz, 1988). While the benefits of modified atmosphere storage are well documented on strawberries (Couey and Wells, 1970; Harvey, 1982a, 1982b), research is sparse and less conclusive on raspberries. Winter et al. (1939) reported increased shelf life of raspberries at 10 to 15C using an initial CO2 concentration of 30%. Smith (1958) found that modified atmospheres with 20% to 25% CO2 slowed ripening and reduced decay. Preharvest applications of fungicides have been shown to control postharvest gray mold occurrence (Freeman and Pepin, 1976). However, the development of fungicide-resistant fungal populations and increasingly stringent governmental regulation limit the use of currently available fungicides. Biological control agents and naturally produced compounds may offer alternative means of controlling fungal infections in raspberries. While little biological control research has been conducted on raspberries, Tronsmo and Dennis (1977) worked with strawberry and found that several microbial organisms, including Trichodenna spp., Gliocladium virens, and Hypocrea semiorbitis, reduced gray mold spoilage in fruit in both the field and storage when applied from early flowering until 2 weeks before harvest. However, postharvest incidence of fruit rot caused by Mucor spp. increased (Janisiewicz and Roitman, 1988). Pyrrolnitrin, isolated from Pseudomonas cepacia, a gramfor publication 7 June 1991. Accepted for publication 27 Sept. 1991. Tire ublishing this paper was defrayed in part by the payment of page Under postal regulations, this paper therefore must be hereby marked ment solely to indicate this fact. r. Soc. Hort. Sci. 117(2):265-270. 1992. negative bacterium, has broad-spectrum antibiotic properties (Imanaka et al., 1965a, 1965b; Janisiewicz and Roitman, 1988). It inhibits growth of fungi, yeasts, and gram-positive bacteria and has low mammalian toxicity (Arima et al., 1965). Takeda et al. (1990) reported inhibition of fungal growth on strawberries treated with a postharvest dip of 250 ppm Pyrrolnitrin and stored at 18C, or at 0C followed by 18C. Unlike some other biocontrol agents, Pyrrolnitrin did not increase the incidence of fruit decay caused by Mucor. The objective of this research was to evaluate the effect of preharvest applications of Pyrrolnitrin on the postharvest longevity of raspberries under various postharvest conditions. Materials and Methods Experiments were conducted in 1989 and 1990 at the Russell E. Larson Research Center in Rock Springs, Pa. For Expts. 1 and 2 (1989), a mature ‘Bristol’ black raspberry planting grown in a hedgerow system according to commercial recommendations was sprayed with recommended rates of captan + benomyl weekly for fungal control until 2 weeks before harvest. At this time, field treatments consisting of 200 ppm Pyrrolnitrin (pure crystals were dissolved in 5 ml of methanol, and distilled water was added for a total volume of 1 liter) plus 0.5 ml Tween 20 wetting agent; captan + benomyl at 1.8 + 0.45 g a.i./liter; or distilled water (control) were applied until drip. Two spray applications were made, on 6 and 13 July 1989. In 1990, ‘Heritage’ red raspberries were harvested from a 3-year-old planting. Fungicide treatments were applied on 13 Sept. For all experiments, berries were harvested 1, 4, and 6 days after treatment application, cooled to 5C immediately after harvest in a forcedair cooler, and kept cold during transport and handling. Each postharvest experimental unit consisted of a 235 x 150 mm plastic tray (a candy box insert) with 24 separate compartments. Individual berries were placed in tray compartments, so that each berry was clearly visible and did not touch the surrounding berries.
