CA2593701A1 - Free fatty acids for interfering with growth of fusarium graminearum - Google Patents

Abstract

The present application discloses methods and compositions for interfering with the growth of the fungus Fusarium graminearum, by exposing the fungus to free fatty acids exhibiting antimicrobial activity. These compositions include compositions comprising the free fatty acids: capric, myistoleic and lauric acids. The application further discloses that these free fatty acids may be derived from cream, such as whey cream, as a virtually unlimited non-limiting source for these molecules.

Description

-2-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.

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.

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 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 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 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 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 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 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.

Claims (11)

We claim:

1. A method for interfering with the growth of Fusarium graminearum comprising exposing the Fusarium graminearum to a composition one or more containing free fatty acids exhibiting antimicrobial activity.

2. The method according to claim 1, wherein the free fatty acids are derived from bovine whey.

3. The method according to claim 1, wherein the free fatty acids comprise capric acid.

4. The method according to claim 1, wherein the free fatty acids comprise myristoleic acids.

5. The method according to claim 1, wherein the free fatty acids comprise lauric acid.

6. The method according to claim 1, where the free fatty acids comprise more than one of capric, myristoleic and lauric acids.

7. Use of one or more free fatty acids exhibiting antimicrobial activity to interfere with the growth of Fusarium Graminearum.

8. The use according to claim 7, wherein the free fatty acids comprise capric acid.

9. The use according to claim 7, wherein the free fatty acids comprise myristoleic acids.

10. The use according to claim 7, wherein the free fatty acids comprise lauric acid.

11. The use according to claim 7, where the free fatty acids comprise more than one of capric, myristoleic and lauric acids.