FREE FATTY ACIDS FOR INTERFERING WITH GROWTH OF FUSARIUM GRAMINEARUM

Field of the Invention

[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 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

Background of the Invention

[0002] 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. Galleπberg. 1997. Scab of wheat and barley: A re-emerging disease of devastating impact. Plant Disease 81:1340-1348)

[0003] Since its emergence, FHB disease has become one of the major factors limiting wheal 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 Fusanum head scab- global status and prospects CIMMYT, Mexico, DJ.; and McMullen, Jones & Gallenberg (1997) supra) During last 15 years, economical losses due to FHB disease were estimated at more than USS3 billion 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 Fusanum Head Blight in wheat and barley Review of Agricultural Economics 26 332- 347; and Wπidels, C E. 2000 Economic and social impacts of Fusanum head blight Changing farms and rural communities in the Northern Great Plains Phytopathology 90.17-21).

[0004] FHB is caused by the Fusanum fungus (Parry, D. W., P Jenkmson, and L. Mcleod.

1995 Fusanum Ear Blight (Scab) in Small-Crain Cereals - A Review. Plant Pathology 44-207-238). Depending on the crop species involved, the regions and the season, several Fusanum species can cause FHB (Parry, et al (1995) supra) However, eptdetnics Within North America are essentially due to F graminearum [telemorphy Gibberella zeae (Schwein) Petch]. FHB infections are initiated when ascopores or macroconidia from Fusanum species land, germinate and penetrate male organs, which stimulates Fusanum 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.

Chnstensen. 1963. Factors affecting resistance of wheat scab caused by Gibbeiella 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 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 speαes 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 ).

[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 of the Invention

[0006] In one aspect of this invention, there is disclosed 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 aαd, launc acid, an unsaturated free fatty and fraction ("UFFA") derived from whey, 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 F. graminearum. The free fatty acids can comprise capric acid, myristoleic acid, lauric and, an unsaturated free fatty aαd fraction ("UFFA") derived from whey, or a combination thereof.

[0008] In another aspect, there is disclosed a F grammearum-resistant seed comprising a seed coated with one or more free fatty acids exhibiting antimicrobial activity.

[0009] In another aspect, there is disclosed 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, myristoleϊc acid, lauric acid, an unsaturated free fatty add fraction ("UFFA") derived from whey, or a combination thereof

Brief Description of the Drawings

[0010] The embodiments of the present disclosure are described below with reference to the accompanying drawings in which:

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 aςids;

Fig. 4B is a photograph showing wheat seeds artifϊdally 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; and

Fig. 5B is a graph showing the percentage of wheat seeds with epicotyl after treatment with different free fatty adds.

Detailed Descriptoin of Preferred Embodiments

[0011] Bovine milk is a natural food ingredient A number of studies have demonstrated that a variety of Free Fatty Acids ("FFA") that arc 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 ot Candida albicans in vitro FEMS Yeast Research 7. 276-2S5). Recent studies have demonstrated that 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)

[0012] 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.

[0013] 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

[0014] While 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

Materials and Reagent

[0015] The whey cieam ("WC") used was obtained from the cheese-maker and milk processor Saputo Inc. (Montreal, Canada). FFA standards were purchased from Slgma-Aldnch All other materials and solvents were of the highest purituy or high-performance HPLC grade (Fisher Scientific).

Strain, media and culture conditions

[0016] The F. graminearum strain (#180378) used was from Dr. Therese Ouellette

(Agriculture and Agri-Food Canada, Ottawa) and was routinely grown m FDA (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 FDA slant agar (0.04% potato starch, 02% dextrose) or Spezieller Nahrstoffarmcr agar (Leslie, J,F. and B.A. Summereli. 2006. The Fusarmm laboratory manual. Blacktvell Publishing).

Examples

[0017] The following examples are included Io demonstrate aspects of the invention It should be appreciated by those of skill in the art that the techniques disclosed m the examples which follow represent techniques that function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the scope of the invention.

Example 1 In vitro antifungal activity of FFA derived from WC

(a) Purification, and fractionation of FFA from WC

[0018] 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 baponificaπon is carried out at 60 C for 60 minutes. The resulting mixture JS then cooled and filtered (40 μm) 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).

[0019] Purified FFA derived from whey cream are then 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 ac ⁱ ds.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 is separated from the urea precipitated by centrifugation (5 min) at 1000 x g. 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 gTavπnetrically. 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).

[0020] This procedure gives three FFA fractions: fraction 1 (Lc unfractioπated EFA), which was likely to contain the majority of saturated, monounsaturated and polyunsaturated fatty acids found in dairy products; fraction 2, containing FFA that do not form inclusion complex with urea; and 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).

(b) In vitro Antifungal Activity

[0021] 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 μg/ml. The antifungal activity was evaluated in 96-weIl microtiter plates (Costar 3595) using PDA liquid media. AU FFA samples were dissolved in ethanol and no more than 1% of ethanol (final concentration) was used in the incubating medium. As negative control, ethanσJ 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% 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 denned 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).

[0022] λs shown m Fig. IA, 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 MIC50 was found to be 113 μg/ml (Table 1). Additional assays showed that antifungal activity against F. graminearum can be exhibited with MIC ₅₀ of UFFA as low as 6

μg/mL The two Other fractions possessed either a weak antifungal activity independent of the concentration or were completely inactive: they were therefore not further investigated.

[0023] The fraction with UFFA was also active when appbed on germinated macroconidia (i.e. 6h post germination in PDA at 25°Q, suggesting that UFPA enriched fraction does not only inhibit the germination of Fusarium macroconidia, but also its development under its active growing form (Fig. IB). Further fractionation on aminopropyl showed that the antifungal activity was associated with FFA present in die UFFA fraction (data not shown)

(c) HPLC fractionation and identification of antifungal compounds [0024] In a subsequent purification step, the UFFA enriched fraction obtained following fractionation on aminopropyl colums was applied on a semi-preparative reverse phase HPLC column HPLC fractionation was performed on a Beckman-Coulter HPLC Gold® system composed of two pumps, a module solvent (model 126), a UV spectrophotometrjc detector (model 168), a fraction collector (SClOO) and a 500-mL sample loop injector (Reodyne 7725ϊ). 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- pak® HR C18, 6 μm, 6θA, 7.S x 300 mm. Waters). UFFA (about 4,5 mg) dissolved in 50% ethanol were applied to the column pre-equϋibrated 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. 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 μl ethanol (75%) and 4 μl of each were tested for antifungal activity.

[0025] 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 m 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 agamst F graminearum (Fig 2B)

[0026] To characterize components of active fractions, commercial reference FFA were analysed by HPLC In this system, the Single peak present in the fraction S was found co-eluted with an identical retention time of capric aαd (C10-0, data not shown). Similarly, fractions 22 and 23 are believed to contain a umque active ingredient as both fractions overlap a single peak co- eluting with mynstoleie acid (C14.1n-5; data not shown).

(d) Mass Spectroscopy

[0027] To characterize constituents further, bioactive fractions were analysed by electrospray ionization mass spectroscopy (ESI/MS). ESI-MS analysis was earned out in negative mode using a MJcromass 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 120 μl/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 (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

[002S] 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:l (mynstoleie add) respectively

[0029] Other molecular ions shown in the fraction 8 and fraction 22/23 (i.e. [M+HCOOH-

H]-, [M ⁺ CH ₃ COOH-H] [- and [2M-HJ-) were also present when commercial preparation of either capnc or mynstoleie acids were subjected to ESl/MS analysis and therefore, are typical of FFA (Fig. 3 and data not shown). Taken together, these observations indicate that the fraction 8 contains capnc aαd (Cl 0:0) while myristoleic acid (C14:1n-5) can be detected in fraction 22/23.

(d) Confirmation of the identity of active compounds inhibiting in vitro growth

[0030] To confirm the antifungal activity of capric and mynstoleic acid, commercial preparations were assayed for their ability to inhibit in vitro the growth of F, graminearum. The mynstoleic add 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. 2001. The composition of bovine milk lipids. January 1995 to December 2CX)O. Journal of Dairy Science 85:295- 350), As shown in Fig. 5, both commercial preparations of capiic or tnyristoleic 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 mynstoleic acids were found to be approximately 34 and 185 μg/ml respectively (Table 1). In addition, the germination of macroconidia was completely mhibited when capric acid was used at concentrations higher than 42 μg/ml (data not shown). This complete inhibition was not alleviated after washing macroconidia with fresh FDA media and prolonged incubation at 25°C, suggesting that the growth inhibition of capric acid on P. 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 mynstoleic acid (da ta not shown).

[0031] To evaluate the specificity of capric and mynstoleic adds, the susceptibility of F. graminearum to a number of PFA present in bovine milk that were not identified in the assay guided fractionation were tested (Jensen (2002), supra). Therefore, antifungal activities of Jauric (12:0), myr-Shc (14.0), palmitoleic (16:ln-7), linoleic (18:2n-6), α-linolenic (18:3n-3), arachidonic (20:4n-6) and oleic (18τln-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 μg/ml (Table 1 and data not shown). It is noteworthy to mention that antifungal activity against the two fungal plant pathogens Rhizoctonia sdani and PytMum ultimum had been previously attributed to lauric acid (Traitlcr et al. (1988), supra). Contraiily to capric acid, a complete inhibition has not been achieved with lauric acid. Therefore, the above results illustrate that the in viirc development of F. graminearum is particularly sensitive to the presence of unsaturated free fatty acids, capric add, lauric acid, mynstoleic add, or a combination of these free fatty acids.

Example 2: In vivo inhibition of F. graminearum growth on artificially infected wheat seeds

[0032] To treat wheat seeds artificially infected with macroconidia of F. graminearum,

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. Preferably, wheat seeds from a local grower, sterilized by an autoclave treatment, 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). F. gramviearum macroconidies (50 macroconidies/10 μl) 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

[0033] Growth of F. graminearum was readily observed after 60 hour incubation when macroconidia were inoculated on the surface of sterilized wheat seeds (Fig. 4A Ctrl) Indeed, after seeds were soaked in either 10 mg/mL of the UFFA enriched fraction or 10 mM of myristoleic acid solution, no gτowth was observed at 60 hours of incubation (Fig. 4A), but the seeds were observed again 7 days after incubation and growth was observed (Pig. 4B). On the other hand, seed treatments with 10 mM of capric aαd inhibit completely the development of F. graminearum, even after 7-days incubation (Fig. 4A and B). Laurie aαd exhibited a poor antifungal activity against F graminearum on slenlized wheat seeds as growth of this fungus was already detected after 6Oh incubation (Fig. 4A) The FFA concentrations used in this seed assay were about 100-fold higher than MIC ₅₀ used in tntro (Table 1)

Example 3: In vivo inhibition of F. graminearum growth on naturally infected wheat seeds

[0034] Wheat seeds used come from naturally infected plants (5602HR FHB Nursery,

Carman, MB) and are not sterilized by autoclaving. To treat wheat seeds naturally infected with Fusanum spp, unsterilized wheat seeds are soaked in 1% whey protein isolate solution containing either 10 mg/ml of the UFFA enriched fraction, 10 mM of capric acid, 10 mM of lauric acid, or 10 mM of myristoleic acid for 10 minutes and then incubated on Malachite Green Agar (MGA) plates for 4Oh at 25°C. Experiments were done in triplicate for each treatment.

[0035] Growth of F. graminearum was observed on all seeds soaked in the carrier solution alone or with lauric or myristoleic acids when the seeds were observed on MGA plates after 40 hours of incubabon (Fig. 5A). However, no or very little growth was observed on seeds soaked in either the UFFA enriched fraction or capric aαd (Fig 5A). Importanlly, the germination of wheat seeds was also observed after 7-days incubation at 25°C and as shown in Fig. 5B, only lauric acid

seems to decrease slightly the germination of wheat seeds in our conditions. This indicates that the viability of wheat seeds is not significantly altered at effective FFA concentrations.