Note: Descriptions are shown in the official language in which they were submitted.
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Free Fatty Acids Agents For Interfering With
Growth of Fusarium Graminearum
Field
[0001] 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 relates to methods and compositions for
interfering with the
growth of Fusarium graminearum, by exposing the fungus to free fatty acids
exhibiting antimicrobial
activity.
Background
[0002] Fusarium graminearum (F. graminearum) is the main causal agent of
Fusarium 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. Gallenberg. 1997. Scab of
wheat and barley: A
reemerging disease of devastating impact. Plant Disease 81:1340-1348).
[0003] Since its emergence, FHB disease has become one of the major factors
limiting wheat
and barley production worldwide, reducing yield by 30-70% (Bai, G. H. and G.
Shaner. 1994. Scab of
Wheat - Prospects for Control. Plant Disease 78:760-766.; Dubin, H. J., L.
Gilchrist, J. Reeves, and A.
McNab. 1997. Fusarium head scab: global status and prospects. CIMMYT, Mexico,
D.F.; and
McMullen, Jones & Gallenberg (1997) supra). During last 15 years, economical
losses due to FHB
disease were estimated at more than 3 billions US$ for wheat and barley
growers (Nganje, W. E., D.
A. Bangsund, F. L. Leistritz, W. W. Wilson, and N. M. Tiapo. 2004. Regional
economic impacts of
Fusarium Head Blight in wheat and barley. Review of Agricultural Economics
26:332-347.; and
Windels, C. E. 2000. Economic and social impacts of Fusariurn head blight:
Changing farms and rural
communities in the Northern Great Plains. Phytopathology 90:17-21.).
[0004] FHB is caused by the Fusarium fungus (Parry, D. W., P. Jenkinson, and
L. Mcleod.
1995. Fusarium Ear Blight (Scab) in Small-Grain Cereals - A Review. Plant
Pathology 44:207-238.).
Depending on the crop species involved, the regions and the season, several
Fusarium species can
cause FHB (Parry, et al. (1995) supra). However, epidemics within North
America are essentially due
to F. graminearum [telemorphy Gibberella zeae (Schwein.) Petch]. FHB
infections are initiated when
ascopores or macroconidia from Fusarium species land, germinate and penetrate
male organs, which
stimulates Fusarium hyphal growth (Adams, J. F. 1921. Observations on wheat
scab in Pennsylvania
and its pathological history. Phytopathology 11:115-124.; Schroeder, H. W. and
J. J. Christensen. 1963.
CA 02593701 2007-07-13
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Factors affecting resistance of wheat scab caused by Gibberella zeae.
Phytopathology 53:831-838.; and
Strange, R. N. and H. Smith. 1978. Effects of choline, betaine and wheat-germ
extract on growth of
cereal pathogens. Trans.Br.Mycol.Soc. 70:193-199.). When infected at early
flowering, susceptible
cereal plants become necrotic, bleached and severely compromised in kernels
development, leading
to important reduction in grain yield (McMullen, Jones & Gallenberg (1997)
supra). In addition,
infected cereal grains are generally unsuitable for food and fed livestock
since several Fusarium
species are toxigenic and produce trichothecene and estrogenic mycotoxins
(Salas, B., B. J. Steffenson,
H. H. Casper, B. Tacke, L. K. Prom, T. G. Fetch, and P. B. Schwarz. 1999.
Fusarium species pathogenic
to barley and their associated mycotoxins. Plant Disease 83:667-674.). There
are safety concern
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 al. (1999)
supra).
[0005] Despite the use of several agricultural strategies and the development
of resistant
cereal plants, completely effective control of FHB disease has still not been
achieved. Attempts to
control FHB with fungicides gave unsatisfactory results since the used of
current fungicides gave
unsatisfactory results (McMullen, M. P., B. Schatz, R. Stover, and T.
Gregoire. 1997. Studies of
fungicide efficacy, application timing, and application technologies to reduce
Fusarium head blight
and deoxynivalenol. Cereal Research Communications 25:779-780.; and
Pirgozliev, S. R., S. G.
Edwards, M. C. Hare, and P. Jenkinson. 2003. Strategies for the control of
Fusarium head blight in
cereals. European Journal of Plant Pathology 109:731-742.). In addition,
continuous application of
chemical fungicides can result in the selection of resistant Fusarium species
and increase public
concern regarding environmental and food contamination with fungicidal
residues recalcitrant to
degradation.
Summary
[0006] In one aspect, there is disclosed a method for interfering with the
growth of Fusarium
graminearum comprising exposing the Fusarium graminearum with a composition
containing free fatty
acids exhibiting antimicrobial activity. The free fatty acids can comprise
capric acid, myristoleic,
lauric acid or a combination thereof.
[0007] In another aspect, there is disclosed a use of a composition containing
free fatty acids
exhibiting antimicrobial activity for interfering with the growth of Fusarium
graminearum. The free
fatty acids can comprise capric acid, myristoleic, lauric acid or a
combination thereof.
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Brief Description of the Drawings
[0008] The embodiments of the present disclosure are described below with
reference to the
accompanying drawings in which:
Fig. 1 illustrates the percentage growth of F. graminearum inhibited by
various concentrations
of differing fractions of free fatty acids;
Fig. 2A illustrates the reverse phase HPLC profile of unsaturated free fatty
acids ("UFFA");
Fig. 2B illustrates the percentage growth of F. graminearum inhibited by
different fractions of
free fatty acids; and
Fig. 3A to C illustrates the major molecular ions of each of the fractions
(ESI-MS).
Detailed Description of Preferred Embodiments
[0009] Bovine milk is a natural food ingredient. A number of studies have
demonstrated
that a variety of Free Fatty Acids ("FFA") that are commonly found in natural
product such as bovine
milk and its derivatives, exhibit antimicrobial activities against several
pathogens including fungi
(Isaacs, C. E. 2001. The antimicrobial function of milk lipids, p. 271-285.).
It has recently been
demonstrated that FFA from bovine whey cream inhibited the germination of the
human fungal
pathogen Candida albicans in vitro (Clement M, Tremblay J, Lange M, Thibodeau
J and Belhumeur P.
2007. Whey derived free fatty acids suppress the germination of Candida
albicans in vitro. FEMS Yeast
Research 7: 276-285.) Recent studies have demonstrated that bovine milk/whey
can be used as a
natural alternative to chemical or toxic fungicides in 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.
Bruer. 2001. Organic control
of powdery mildew without sulfur. Australian Grapegrower and Winemaker
452:22.).
[00010] The application discloses that a fraction enriched in UFFA display a
potent in vitro
antifungal activity against F. graminearum. Further separation by HPLC led to
the identification of
capric and myristoleic acids as the primary antifungal components of UFFA from
bovine whey.
1. Materials and Methods.
Materials and Reagent
[00011] The whey cream ("WC") used was obtained from the cheese-maker and milk
processor Saputo Inc (Montreal, Canada). FFA standards were purchased from
Sigma-Aldrich. All
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other materials and solvents were of the highest purity or high-performance
HPLC grade (Fisher
Scientific).
Strain, media and culture conditions
[00012] The F. graminearum strain (#180378) used in this study was from Dr.
Therese
Ouellette (Agriculture and Agri-Food Canada, Ottawa) and was routinely grown
in PDA (0.4 %
potato starch, 2 % dextrose; Difco) medium at 25 C. Macroconidia of F.
graminearum were obtained
after 5 days at 25 C on diluted PDA slant agar (0.04 % potato starch, 0.2 %
dextrose).
Free Fatty Acids Purification
[00013] Total lipids of WC were 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). Saponification of WC was 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 saponification was carried out at 60 C for 60 minutes. The resulting
mixture was cooled
and filtered (40 pm) to remove solids. The solution was acidified to pH 1 with
HCl-H201:1 (v/v) and
lipids were extracted three times with hexane (40 ml). Pooled hexane fractions
were neutralized by
washing with water and then dried under nitrogen. FFAs were recovered by
extraction on an
aminopropyl disposable column as describe previously (Clement, et al. (2007),
supra).
Urea Fractionation
[00014] FFA derived from whey cream were fractionated by the means of the urea
inclusion
procedure (Traitler, H., H. J. Wille, and A. Studer. 1988. Fractionation of
Blackcurrant Seed Oil.
Journal of the American Oil Chemists Society 65:755-760.). The ratio of fatty
acids:urea was 1:4 (w/w)
and the ratio of urea:methanol was 1:3 (w/v). After completion of
crystallization at a final
temperature of 2 C, the methanol phase enriched in UFFA was separated from the
urea precipitated
by centrifugation (5 min) at 1000 x g. The urea crystals enriched in saturated
free fatty acids ("SFFA")
were washed once with urea saturated methanol to improve the yield of the UFFA
enriched fraction.
UFFA and SFFA were recovered as described (Traitler, et al. (1988), supra) and
dried under nitrogen.
Yields were determined gravimetrically. The efficiency of the urea
fractionation was evaluated by
HPTLC (Clement, et al. (2007), supra) since, as compared to SFFA, UFFA appear
red after revelation
with sulfuric acid (data not shown).
Fractionation by HPLC
[00015] HPLC fractionation was performed on a Beckman-Coulter HPLC Gold
system
composed of two pumps, a module solvent (model 126), a UV spectrophotometric
detector (model
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168), a fraction collector (SC100) and a 500-mL sample loop injector (Reodyne
7725i). The recorded
HPLC spectra were analyzed using the 32 karaf software (Beckman-Coulter). The
UFFA enriched
fraction was separated by reverse-phase HPLC on a semi-preparative C18 column
(Prep Nova-pak0
HR C18, 6 m, 60A, 7.8 x 300 mm, Waters). UFFA (about 4.5 mg) dissolved in 50
% ethanol were
applied to the column pre-equilibrated in 50 % acetonitrile: 0.1 % TFA and
eluted by a linear gradient
to 100 % acetonitrile: 0.1 % TFA from 0 to 70 min at a flow rate of 8 ml/min.
UV detection was used
to monitor effluent at 215 nm. Water (10 ml) was added to each collected
fractions and then extracted
three times with hexane. After drying under nitrogen, each fraction was
reconstituted in 30 l ethanol
(75 %) and 4 l of each were tested for antifungal activity.
Biological Testing
[00016] The antifungal activity was evaluated in 96-well microtiter plates
(Costar 3595) using
PDA liquid media. All FFA samples were dissolved in ethanol and no more than 1
% of ethanol (final
concentration) was used in the incubating medium. As negative control, ethanol
without sample was
used and applied to fungi culture under the same experimental conditions.
Macroconidia of F.
graminearum were harvested by washing vigorously the slant cultures with 5 ml
of 0.9 % NaCI.
Coarse debris were removed by filtration through a sterile cotton plugs
inserted into a Pasteur pipet.
Using an hemacytometer, the cell suspensions were adjusted in PDA medium at a
concentration of 5
x 103 macroconidia/ml. Microtiter 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 250C in atmospheric incubators during
39 h. The minimal
inhibitory concentration (MICso) was defined as the lowest FFA concentration
reducing by 50 % the
optical density at 630 nm 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 optical density determination. The spectrophotometer used
was from Dynatech
(MicroplateOReader MR600).
Mass Spectroscopy
[00017] ESI-MS analysis was carried out in negative mode using a Micromass
Quattro II
Triple Quadrupole Mass Spectrometer equipped with an electrospray source.
Samples dissolved in
isopropanol 50% containing 25mM triethylamine were infused at a flow rate of
1201z1/h. Data were
accumulated in MCA mode for one minute and analyses were carried out using
MassLynx version
3.5 software. Nitrogen was used as curtain gas (400 1/h ) and nebulising gas
(20 1/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
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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.
II. Results and Discussion
Purification and fractionation of FFA from WC
[00018] Frozen whey cream from Saputo Inc. was first extracted by the method
of Bligh and
Dyer (Bligh & Dryer (1959), supra). After evaporation to dryness, extracted
lipids were subjected to
saponification and the resulting FFA were purified by solid phase extraction
using an aminopropyl
column (Clement, et al. (2007), supra). Purified FFA were then fractionated by
the urea inclusion
procedure (Traitler, et al. (1988), supra). This procedure gives three FFA
fractions: the fraction 1 (i.e.
unfractionated FFA), which was likely to contain the majority of saturated,
monounsaturated and
polyunsaturated fatty acids found in dairy products; the fraction 2, FFA that
do not form inclusion
complex with urea; the fraction 3. FFA that form complex with urea. Analysis
by HPTLC revealed
that as expected the fraction 2 was enriched in UFFA while SFFA were found in
fraction 3 (data not
shown).
Antifungal activity of FFA derived from WC
[00019] An aliquot of the above fractions was tested for antifungal activity
against F.
graminearum. To evaluate the effect of concentration on the germination of
macroconidia, each
fraction was tested in triplicate at concentrations of 0, 12.5, 25, 50, 100
and 200 }rg/ml. As shown in
Fig. 1, only the fraction enriched in UFFA exhibited an antifungal activity
against F. graminearum.
This activity was dependent on the concentration and using dose-response
curves, the MICs0 was
found to be 113 pg/ml (Table 1). The two other fractions possessed either a
weak antifungal activity
independent of the concentration or were completely inactive: they were
therefore no further
investigated (Fig. 1).
Table 1. In vitro antifungal activity of FFA against F. graminearum
Samples MICs0 (ug/ml)
FFA >200
UFFA 113 3.79
SFFA >200
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Table 1. In vitro antifungal activity of FFA against F. graminearum
Samples MIC50 (ug/ml)
Capric (C 10:0) 34 1.11
Myristoleic (C 14:1n-5) 185 4.15
Lauric (C 12:0) 132 3.42
HPLC fractionation and identification of antifungal compounds
[00020] In a subsequent purification step, the UFFA enriched fraction was
applied on a semi-
preparative reverse phase HPLC column. Compounds eluting with increasing
acetonitrile
concentrations were detected using a Photodiode Array ("PDA") detector. As
shown in the Fig. 2A,
several peaks were detected using a wavelength of 215 nm, indicating that the
UFFA enriched
fraction was complex in composition. Fractions corresponding to each peak were
collected and tested
for their ability to inhibit in vitro the growth of F. graminearum. Despite
the high complexity of the
UFFA enriched fraction, only fraction 8, 22 and 23 were found to exhibit a
significant antifungal
activity against F. graminearum (Fig. 2B).
[00021] To characterize components of active fractions, commercial reference
FFA were
analysed by HPLC. In this system, the single peak present in the fraction 8
was found to co-eluted
with an identical retention time of capric acid (C10:0, data not shown).
Similarly, fractions 22 and 23
are believe to contain a unique active ingredient as both fractions overlap a
single peak co-eluting
with myristoleic acid (C14:1n-5; data not shown).
[00022] To characterize constituents further, bioactive fractions were
analysed by
electrospray ionization mass spectroscopy (ESI/MS). As shown in Fig. 3, major
molecular ions [M-
H]- for the fraction 8 and fractions 22/23 were respectively m/z of 171 and
225. Based on the sum of
carbon, hydrogen an oxygen number, these molecular ions were found to
correspond to C10:0 (capric
acid) and C14:1 (myristoleic acid) respectively.
[00023] Other molecular ions shown in the fraction 8 and fraction 22/23 (i.e.
[M+HCOOH-
H]-, [M'CH3COOH-H] [- and [2M-H]-) were also present when commercial
preparation of either
capric or myristoleic acids were subjected to ESI/ MS analysis and therefore,
are typical of FFA (Fig. 3
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and data not shown). Taken together, these observations indicate that the
fraction 8 contains capric
acid (C10:0) while myristoleic acid (C14:1n-5) can be detected in fraction
22/23.
Confirmation of the identity of active compounds
[00024] To confirm the antifungal activity of capric and tetradecenoic acid,
commercial
preparations were assayed for their ability to inhibit in vitro the growth of
F. graminearum. The
myristoleic acid isomer (14:ln-5) was used since as mentioned above, this
fatty acid was found to
elute from the HPLC column with a retention time identical to the peak of
fractions 22 and 23. In
addition, bovine milk only contains the 9-myristoleic acid isomer (Jensen, R.
G. 2002. The
composition of bovine milk lipids: January 1995 to December 2000. Journal of
Dairy Science 85:295-
350.). As shown in Fig. 5, both commercial preparations of capric or
myristoleic acids were active at
inhibiting the growth of F. graminearum. The activity of these FFA was
dependent of the
concentration and using dose-response curves, MIC50 for capric and myristoleic
acids were found to
be approximately 34 and 185 }zg/ml respectively (Table 1). In addition, the
germination of
macroconidia was completely inhibited when capric acid was used at
concentrations higher than 42
}rg/ml (data not shown). This complete inhibition was not alleviated after
washing macroconidia
with fresh PDA media and prolonged incubation at 250C, suggesting that the
growth inhibition of
capric acid on F. graminearum was irreversible (data not shown). Such
inhibition could suggest that
capric acid is fungicidal to macroconidia of F. graminearum. Contrary to
capric acid, a complete
inhibition was not possible with myristoleic acid (data not shown).
[00025] To evaluate the specificity of capric and myristoleic acids, the
susceptibility of F.
graminearum to a number of FFA present in bovine milk that were not identified
in the assay guided
fractionation were tested (Jensen (2002), supra). Therefore, antifungal
activities of lauric (12:0),
myristic (14:0), palmitoleic (16:ln-7), linoleic (18:2n-6), a-linolenic (18:3n-
3), arachidonic (20:4n-6) and
oleic (18:ln-9) acids were evaluated. None of these FFA exhibited an in vitro
antifungal activity
against F. graminearum, except for lauric acid which reduced by 50 % the
development of F.
graminearum at 132 pg/ml (Table 1 and data not shown). Contrarily to capric
acid, a complete
inhibition has not been achieved with lauric acid. Therefore, the above
results illustrate that the in
vitro development of F. graminearum is particularly sensitive to the presence
of capric, lauric or
myristoleic acids.
[00026] The present application illustrates that bovine whey contains
bioactive FFA that can
inhibit the growth of F. graminearum. Despite the fact that bovine milk/whey
contains more than
four hundred different fatty acids (Jensen (2002), supra), the assays isolated
only capric and
myristoleic acids. In vitro assays with commercial preparations of several
FFAs reported to be
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present in bovine milk confirmed that only capric and myristoleic acids
exhibited antifungal activity
against F. graminearum (Table 1).
[00027] Recently, an in vitro antifungal activity against the fungal plant
pathogenic fungi
Rhizoctonia solani and Pythium ultimum was attributed to lauric acid (Walters,
D. R., R. L. Walker,
and K. C. Walker. 2003. Lauric acid exhibits antifungal activity against plant
pathogenic fungi.
Journal of Phytopathology-Phytopathologische Zeitschrift 151:228-230.).
Similarly, we found that
lauric acid was also active in vitro against F. graminearum (MIC50 132 pg/ml).
Nevertheless, lauric acid
was not isolated from the assay-guided fractionation of the present
application, even if this fatty acid
is abundant in bovine milk (Jensen (2002), supra). Since lauric acid is more
efficiently precipitated
than capric acid by urea (data not shown), this suggests that lauric acid is
likely to be preferentially
enriched in the SFFA fraction rather than in the UFFA fraction (data not
shown). Indeed, lauric acid is
not found in the UFFA enriched fraction as judged by the retention time (i.e.
14.4 minutes) of
commercial preparation of lauric acid on the C18 HPLC column (Fig. 2A and data
not shown).
Therefore, even if the SFFA enrich fraction did not display a strong
antifungal activity against F.
graminearum (Fig. 1), lauric acid as one of its purified component could.
[00028] 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.
[00029] While bovine milk is a source for the free fatty acids discussed
herein, it will be
understood by those skilled in the art that other sources for the free fatty
acids can be used or
alternatively, the free fatty acids can be synthesized chemically.
