WO2009009885A1 - Acides gras libres interférant avec la croissance de fusarium graminearum - Google Patents

Acides gras libres interférant avec la croissance de fusarium graminearum Download PDF

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Publication number
WO2009009885A1
WO2009009885A1 PCT/CA2008/001292 CA2008001292W WO2009009885A1 WO 2009009885 A1 WO2009009885 A1 WO 2009009885A1 CA 2008001292 W CA2008001292 W CA 2008001292W WO 2009009885 A1 WO2009009885 A1 WO 2009009885A1
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WIPO (PCT)
Prior art keywords
free fatty
fatty acids
graminearum
effective amount
acid
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PCT/CA2008/001292
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English (en)
Inventor
Martin CLÉMENT
Jessy Tremblay
Michel Lange
Jacques Thibodeau
Pierre Belhumeur
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Université de Montréal
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Publication of WO2009009885A1 publication Critical patent/WO2009009885A1/fr

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    • 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
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof
    • 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
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/06—Unsaturated carboxylic acids or thio analogues thereof; Derivatives thereof
    • 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
    • A01N63/10—Animals; Substances produced thereby or obtained therefrom

Definitions

  • the present disclosure relates to methods and compositions for interfering with the growth of fungi by exposing the fungi to free fatty acids exhibiting antimicrobial activity More particularly, the present disclosure Telates to methods and compositions for interfering with the growth of Fusarium graminearum (F. graminearum), by exposing the fungus to free fatty acids exhibiting antimicrobial activity
  • F. graminearum Fusarium graminearum
  • F. graminearum is the main causal agent of Fusanum head blight disease ("FHB"), a disease of small cereal grains FHB has emerged as one of the most economically devastating fungal disease of small cereal grains in growing regions of Canada, U.S.A., China and Europe (McMullen, M , R. Jones, and D. Galle ⁇ berg. 1997. Scab of wheat and barley: A re-emerging disease of devastating impact. Plant Disease 81:1340-1348)
  • FHB is caused by the Fusanum fungus (Parry, D. W., P Jenkmson, and L. Mcleod.
  • infected cereal grains are generally unsuitable for food and fed livestock since several Fusarium species are toxigenic and produce trichotheeene and estrogenic mycotoxins (Salas, B., B. J. Steffenson, H. K. Casper, B. Tacke, L. K. Prom, T. G. Fetch, and P. B. Schwarz. 1999. Fusarium spe ⁇ es pathogenic to barley and their associated mycotoxins. Plant Disease 83:667-674). There are safety concerns regarding mycotoxins produced by some Fusarium species as they can accumulate to non-negligible levels in infected tissues and can cause serious hazard to animal and plant health (Salas, et id. (1999) supra ).
  • a method for interfering with the growth of F. graminearum comprising exposing the F, graminearum with a composition containing free fatty acids exhibiting antimicrobial activity.
  • the free fatty acids can comprise capric acid, myristoleic a ⁇ d, launc acid, an unsaturated free fatty and fraction ("UFFA") derived from whey, or a combination thereof
  • a composition containing free fatty acids exhibiting antimicrobial activity for interfering with the growth of F. graminearum.
  • the free fatty acids can comprise capric acid, myristoleic acid, lauric and, an unsaturated free fatty a ⁇ d fraction ("UFFA") derived from whey, or a combination thereof.
  • UFFA unsaturated free fatty a ⁇ d fraction
  • a F grammearum-resistant seed comprising a seed coated with one or more free fatty acids exhibiting antimicrobial activity.
  • a method of interfering with the growth of F- graminearum on a seed comprising the steps of coating the seed with one or more free fatty acids exhibiting antimicrobial activity.
  • the free fatty acids can comprise capric acid, myristole ⁇ c acid, lauric acid, an unsaturated free fatty add fraction ("UFFA") derived from whey, or a combination thereof
  • Fig. IA is a graph showing the percentage growth of F. graminearum inhibited by various concentrations of differing fractions of free fatty acids
  • Fig. IB is a graph showing the percentage growth of germinated and ungerminated F. graminearum inhibited by the unsaturated free fatty acids fraction;
  • Fig. 2A is a graph showing the reverse phase HPLC profile of UFFA
  • Fig. 2B is a graph showing the percentage growth of F. graminearum inhibited by different FtFLC fractions of free fatty acids in vitro;
  • Fig.3A is a graph showing the major molecular ions of fraction 8 of Fig. 2B (ESI-MS);
  • Fig.3B is a graph showing the major molecular ions of fraction 22 of Fig. 2B (ESl-MS),
  • Fig. 3C is a graph showing the major molecular ions of fraction 23 of Fig. 2B (ESl-MS);
  • Fig. 4A is a photograph showing wheat seeds artificially inoculated With macroconidia of F graminearum observed 60 hours after having been treated with different free fatty a ⁇ ids;
  • Fig. 4B is a photograph showing wheat seeds artif ⁇ dally inoculated with macroconidia of F. graminearum observed 7 days after having been treated with different free fatty acids;
  • Fig. 5A is a photograph showing wheat seeds naturally infected with Fusarium spp. observed 40 hours after having been treated with different free fatty acids;
  • Fig. 5B is a graph showing the percentage of wheat seeds with epicotyl after treatment with different free fatty adds.
  • Bovine milk is a natural food ingredient
  • FFA Free Fatty Acids
  • bovine milk/whey can be used as a natural alternative to chemical or toxic fungicides m organic agriculture (Bettiol, W. 1999 Effectiveness of cow's milk against zucchini squash powdery mildew (Sphaerotheca fuliginea) in greenhouse conditions. Crop Protection 18:489-492 ; and Crisp, P and D Eruer 2001 Organic control of powdery mildew without sulfur Australian Grapegrower and Wmemaker 452-22)
  • the application discloses that a fraction enriched in UFFA display a potent in vitro and in vivo antifungal activity against F graminearum in both the germinated and ungemunated states. Further separation by HPLC led to the identification of capiic and mynstoleic acids as the primary antifungal components of UFFA from bovme whey. The application also discloses that while UFFA from bovine whey and caprie acid do not significantly affect the viability of naturally infected wheat seeds, they exhibit antifungal activity against F graminearum.
  • Bovine whey is produced annually in large volumes by the dairy industry .
  • the finding that bovine whey contains FFA with antifungal activity against the cereal fungal pathogen F graminearum suggests that bovine whey or specific component of it could become a virtually unlimited source of these FFA molecules for natural antifungal agents against FHB disease
  • bovme milk is a source for the free fatty acids discussed herein, it will be understood by those skilled in the art mat other sources for the free fatty acids can be used or alternatively, the free fatty acids can be synthesized chemically
  • WC whey cieam
  • Total lipids of WC from Saputo Inc. is first extracted by the Bligh and Dyer procedure (Bligh, E. G. and W. J. Dyer 1959 A rapid method of total lipid extraction and purification. Can.J.Med.Sci. 37.911-917; Clement, et al (2007), supra). After evaporation to dryness, extracted lipids were subjected to saponification performed in a 100 mL glass beaker covered with aluminium foil. Typically, 1 g of WC was mixed with 76 ml of ethanol (96%) containing 1 6 g of KOH. The baponifica ⁇ on is carried out at 60 C for 60 minutes.
  • the resulting mixture JS then cooled and filtered (40 ⁇ m) to remove solids using an aminOpropyl column (Clement, et al. (2007), supra)
  • the solution was acidified to pH J with HCl-H 2 O 1.1 (v/v) and lipids were extracted three times with hexane (40 ml). Pooled hexanc fractions were neutralized by washing with water and then dried under nitrogen. FFAs were recovered by extraction On an aminopropyl disposable column (Clement, et al (2007), supra).
  • the urea crystals enriched in saturated free fatty acids (“5FFA”) are washed once with urea saturated methanol to improve the yield of the UfFA enriched fraction.
  • UFFA and SFFA are recovered as described (Trairler, et al (1988), supra) and dried under nitrogen. Yields can be determined gTav ⁇ netrically.
  • the efficiency of the urea fractionation can be evaluated by HFTLC (Clement, et al. (2007), supra) since, as compared to SFFA, UFFA appear red after revelation with sulfuric acid (data not shown).
  • fraction 1 Lc unfractio ⁇ ated EFA
  • fraction 2 containing FFA that do not form inclusion complex with urea
  • fraction 3 containing FFA that form complex with urea.
  • Analysis by HPTLC revealed that fraction 2 was enriched in UFFA (as well as most unsaponifiable lipids such as cholesterol) while SFFA were found in fraction 3 (data not shown).
  • graminearum were harvested by washing vigorously the slant cultures with 5 ml of 0.9% NaCl. Coarse debris were removed by filtration through sterile cotton plugs inserted into a Pasteur pipette. Using a hemacytometer, the cell suspensions were adjusted in PDA medium at a concentration of 5 x 10 3 macroconidia/ ml Micro titer wells containing 0.1 ml of PDA liquid media supplemented with different concentrations of FFA were inoculated with 0.1 ml of the homogenous macroconidia suspension. The trays were incubated at 25°C in atmospheric incubators during 39 h.
  • the minimal inhibitory concentration (MIC 50 ) was ed as the lowest FFA concentration reducing by 50% the optical density at 630 nim of samples to sample-free control. To avoid interference of the cloudy appearance of some FFA preparations in incubation media, wells were washed three times with PDA liquid media before opbcal density determination.
  • the spectrophotometer used was from Dynatech (MicroplateOReader MR60Q).
  • the UFFA enriched fraction was separated by reverse-phase HPLC on a semi-preparative C18 column (Prep Nova- pak® HR C18, 6 ⁇ m, 6 ⁇ A, 7.S x 300 mm. Waters).
  • UFFA about 4,5 mg
  • 50% ethanol were applied to the column pre-equ ⁇ ibrated in 50% acetonitnle- 01 % TFA and ehited by a linear gradient to 100% acetonitrile.
  • 0.1% TFA from 0 to 70 mm at a flow rate of 8 ml/ mm UV detection was used to monitor effluent at 215 ran.
  • ESI/MS electrospray ionization mass spectroscopy
  • Nitrogen was used as curtain gas (400 1/h) and nebulising gas (201/h)
  • the ESI capillary was set at 2.5 kV while the MS analysis was carried out at a cone voltage of 25 V, a scan rate of 300 Da/s with an inter-scan delay of 0.1 s and a scan range of 135-500 Da.
  • the resolving power was set to obtain unit resolution
  • Example 2 In vivo inhibition of F. graminearum growth on artificially infected wheat seeds
  • FFA dissolved in ethanoi are first transferred to glass tubes. After solvent evaporation under nitrogen, FFA ar e then dissolved in 30 pi of ethane! and immediately, 3 ml of a carrier solution (1 % Whey Protein Isolate from Saputo Inc.) containing 10 mg/ml of the UFFA enriched fraction, 10 mM of capric acid, 10 mM of lauric acid, or 10 mM of myristoleic add was added followed by a brief sorucation treatment (5 sec) m a water bath sonicator (Branson 1210 Bransonic, Danbury, CT) to ensure emulsification of FFA.
  • a carrier solution (1 % Whey Protein Isolate from Saputo Inc.
  • wheat seeds from a local grower are then soaked in the FFA-camer emulsion for 10 minutes and then transferred on a 2% agar plate (Terzi, V , Morcia, C, Faccioli, P., Valè, G , Tacconi, G and Malnati, M. (2007).
  • In vitro antifungal activity of the tea tree (Melaleuca alternifolia essential oil and its major components against plant pathogens. Lett Appl Microbiol. 44(6):613-8).
  • gramviearum macroconidies (50 macroconidies/10 ⁇ l) can then be applied on seeds and the plate incubated for 60h at 25°C Experiments should be performed at least in triplicate for each treatment, As a control, 1 % ethanol without FFA and a carrier solution was used. The method is also repeated with incubation for 7 da ys at 25°C
  • Example 3 In vivo inhibition of F. graminearum growth on naturally infected wheat seeds

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Dentistry (AREA)
  • Agronomy & Crop Science (AREA)
  • Environmental Sciences (AREA)
  • Pest Control & Pesticides (AREA)
  • Plant Pathology (AREA)
  • Wood Science & Technology (AREA)
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Abstract

La présente invention décrit des procédés et des compositions permettant d'interférer avec la croissance du champignon Fusarium graminearum par exposition du champignon à des acides gras libres présentant une activité antimicrobienne. Ces compositions comprennent des compositions incluant des acides gras libres comme un acide gras insaturé, un acide caprique, un acide myristoléïque et un acide laurique. De plus, la présente invention indique que ces acides gras libres peuvent provenir de la crème, du lactosérum par exemple, qui constitue une source non limitante et pratiquement illimitée de ces molécules.
PCT/CA2008/001292 2007-07-13 2008-07-14 Acides gras libres interférant avec la croissance de fusarium graminearum WO2009009885A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CA2,593,701 2007-07-13
CA002593701A CA2593701A1 (fr) 2007-07-13 2007-07-13 Acides gras libres permettant de contrecarrer la croissance du fusarium graminearum

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021235609A1 (fr) * 2020-05-20 2021-11-25 영남대학교 산학협력단 Composition antibactérienne et inhibitrice de la formation de biofilm contenant de l'acide myristoléique en tant que principe actif

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07320936A (ja) * 1994-05-24 1995-12-08 Taiyo Yuden Co Ltd 積層形チップインダクタ
WO1996028022A2 (fr) * 1995-03-08 1996-09-19 Mycogen Corporation Compositions a base d'acides gras et procedes de protection contre les infections des plantes et de lutte antiparasitaire
WO2001097799A1 (fr) * 2000-06-20 2001-12-27 Nutrition Sciences Acides gras a chaines moyennes utilisables comme agents antimicrobiens
US20040266852A1 (en) * 2002-01-09 2004-12-30 Coleman Robert D. Fungicide compositions

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07320936A (ja) * 1994-05-24 1995-12-08 Taiyo Yuden Co Ltd 積層形チップインダクタ
WO1996028022A2 (fr) * 1995-03-08 1996-09-19 Mycogen Corporation Compositions a base d'acides gras et procedes de protection contre les infections des plantes et de lutte antiparasitaire
WO2001097799A1 (fr) * 2000-06-20 2001-12-27 Nutrition Sciences Acides gras a chaines moyennes utilisables comme agents antimicrobiens
US20040266852A1 (en) * 2002-01-09 2004-12-30 Coleman Robert D. Fungicide compositions

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HAMZEHZARGHANIA H. ET AL.: "Metabolic profiling and factor analysis to discriminate quantitative resistance in wheat cultivars against fusarium head blight", PHYSIOLOGICAL AND MOLECULAR PLANT PATHOLOGY, vol. 66, no. 4, April 2005 (2005-04-01), pages 119 - 133 *
PATENT ABSTRACTS OF JAPAN *
SHERWOOD R.F. ET AL.: "Production of the mycotoxin, zearalenone, by Fusarium graminearum growing on stored grain. II. Treatment of wheat grain with organic acids", JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, vol. 25, no. 9, 1974, pages 1089 - 1093 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021235609A1 (fr) * 2020-05-20 2021-11-25 영남대학교 산학협력단 Composition antibactérienne et inhibitrice de la formation de biofilm contenant de l'acide myristoléique en tant que principe actif

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