Pesticide Biochemistry and Physiology 108 (2014) 21–26

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Pesticide Biochemistry and Physiology

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Inhibition of Fusarium graminearum growth and mycotoxin productionby phenolic extract from Spirulina sp.

0048-3575/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.pestbp.2013.11.002

⇑ Corresponding author. Fax: +55 5332338645.E-mail addresses: nandapagnu@terra.com.br, fapagnussatt@furg.br

(F.A. Pagnussatt).

Fernanda Arnhold Pagnussatt a,⇑, Emerson Medeiros Del Ponte b, Jaqueline Garda-Buffon a,Eliana Badiale-Furlong a

a Escola de Química e Alimentos, Universidade Federal do Rio Grande, Rio Grande, RS, Brazilb Departamento de Fitossanidade, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

a r t i c l e i n f o

Article history:Received 16 June 2013Accepted 22 November 2013Available online 1 December 2013

Keywords:Gibberella zeaePhenolic acidNatural fungicideMycelial growthGlucosamineMycotoxins

a b s t r a c t

Fusarium graminearum is a fungal species complex pathogenic occurring worldwide, mainly associatedwith cereal crops. The most important Fusarium mycotoxins are fumonisins, zearalenone and trichothec-enes. The availability of efficient control measures that are less harmful to both the environment and theconsumers is urgent. For such, phenolic acids (PAs) from natural sources are known to reduce fungal con-taminations. This work aimed to identify the PAs present in a culture extract of Spirulina algae (strainLEB-18) and evaluate its effect on mycelial growth rate, glucosamine level, amylase activity and myco-toxin production by four strains of two lineages of F. graminearum. Results showed that amendment ofpotato dextrose media with LEB-18 extract (3% w/v), which was mainly composed by gallic acid, greatlyreduced radial growth of fungal colonies compared to media containing a single PA and the control. Also,average reductions of 40% and 62% in the glucosamine levels and the amylase activity were observed. Ingeneral, the LEB-18 extract and the PAs reduced mycotoxin concentration, with an average reduction of68% for the trichothecene mycotoxins deoxynivalenol and nivalenol.

� 2013 Elsevier Inc. All rights reserved.

1. Introduction

Several Fusarium spp. are causal agents of plant diseases thatlead to significant quantitative losses in agricultural crops and posea threat and food safety especially due to their ability to producemycotoxins of toxicological implications for humans and animals[1,3]. Among the several Fusarium mycotoxins, trichothecenes[deoxynivalenol (DON) and nivalenol (NIV)], fumonisins and zea-ralenone are of major relevance for cereal crops [2,4]. Fusariumgraminearum is the main cause of Fusarium head blight (FHB) dis-ease in wheat and barley and Gibberella ear rot in corn. F. graminea-rum was recently separated into lineages and names were given tophylogenetic species based on genealogical concordance [5].Although morphologically indistinct, these lineages or species varyin relation to aggressiveness, toxigenic potential and geographicdistribution [1,6]. Two phylogenetic species of the complex: F.graminearum and Fusarium meridionale are the two most dominantspecies affecting wheat and barley in Brazil and PCR-based assaysidentified the former possessing a 15-acetyldeoxinivalenol (15-ADON) genotype, and the later possessing a NIV genotype [7–10].

The use of integrated management tactics, such as the use ofresistant cultivars and fungicide sprays during flowering are themost effective measures, but losses in both the yield and qualityof grain cannot be prevented under environmental conditionsfavorable for epidemics [6]. Alternatives to chemical fungicides,such as biocontrol agents (BCAs), have been tested extensively inboth the greenhouse and field environment but the FHB controlefficacy under field conditions has not been consistent [11].Although phenolic compounds from plants and microalgae exhib-ited anti-fungal and anti-mycotoxigenic effects [12,14], the under-lying mechanisms leading to those effects remain to be betterunderstood.

As a product of the secondary metabolism, phenolic compoundsare derived from the glucose metabolism by different biochemicalreactions, which include the shikimic acid or acetate pathways[15]. These compounds can be found in nature in their free, bondedor polymeric forms, and are characterized by the aromatic struc-ture associated to carboxylic groups and one or more hydroxyland/or methyl groups [16]. These compounds can be classified asflavonoids, cummarins and phenolic acids; being the former themost abundant [17].

The presence of multiple phenolic acids in crude extracts of dif-ferent sources may lead to a synergistic effect on fungal growthinhibition. Thus, the identification of the major compounds andtheir evaluation either singly or in mixture is a critical step to

Table 1Fusarium graminearum species complex strains examined in this study.

Phylogenetic speciesa Code Host oforigin

Trichothecenegenotypeb

Fusarium graminearum s.str.

07Tr001 Wheat 15-ADON

22 F.A. Pagnussatt et al. / Pesticide Biochemistry and Physiology 108 (2014) 21–26

understand and evaluate the potential antifungal properties ofphenolic acids. Therefore, this study aimed to evaluate the effectsof phenolic acids, either in their purified or in a crude extract fromcultures of Spirulina sp., on mycelial growth, glucosamine content,enzyme activity and mycotoxin production of pathogenic F. grami-nearum strains.

Fusarium graminearum s.str.

08Tr024 Wheat 15-ADON

Fusarium meridionale 07Tr025 Wheat NIVFusarium meridionale 07Cv022 Barley NIV

a According to O’Donnell et al. [5] and identified in previous studies (Astolfi et al.,[7,10]).

b Determined based on portions of Tri3 and Tri12 genes using a PCR assay (Astolfiet al., [7,10]).

2. Materials and methods

2.1. Biomass production, drying, extraction and quantification ofphenolic compounds

Spirulina sp. strain (LEB-18) was grown in tanks containingwater (taken from the Lagoa Mangueira lagoon) supplementedwith 20% (v/v) of Zarrouk medium. This were referred as MLW-Sin glass fiber tanks, agitated by paddles located at the bottom ofthe tank. The algae biomass was separated by filtration after reach-ing a concentration of 1 g L�l. The filtrate was extracted, dried outin a tray dryer at 50 �C for 5 h, vacuum packed and stored in a coldchamber [18]. The dry biomass was ground to 32 mesh and the freephenolic compounds were extracted with methanol [13] and quan-tified (Folin–Ciocalteau method) using an analytical curve of gallicacid (4–50 lg ml�1).

2.2. Profile of phenolic acids

The chromatographic standards (98–99% purity) used for thedetermination of the phenolic acids were gallic acid, caffeic acid,salicylic acid and trans-cinnamic acid (Fig. 1) (Sigma ChemicalsCo. Methanol, HPLC grade, Merck) and 0.1 mg ml�1 stock solutionswere prepared for each acid. Separation, identification and quanti-fication of the standards acids and those of the LEB-18 extract wereperformed in a high performance liquid chromatography (HPLC)with UV–Vis detector 10AXL, equipped with a reverse phase col-umn RP-18 CLC-ODS octadecyl (5 lm � 4.6 � 150 mm). The gradi-ent elution was carried out using an acetic acid aqueous solution(99:1, v/v), methanol and acetonitrile as mobile phase, with a flowrate of 1 ml min�1. Eighteen minutes were necessary to elute allthe standards.

2.3. Fungal isolates and initial culturing conditions

The fungal isolates used in this study were selected based ontheir known aggressiveness and varying toxigenic potential deter-mined in previous studies. They differed according to the host oforigin, phylogenetic species and trichothecene genotype and repre-sent the most dominant species causing head blight in wheat andbarley in Brazil [6,7,10] (Table 1).

The fungal isolates were grown on SNA (Spezieller Nährstoffarm-er Agar) at 25 �C and mycelial plugs were maintained at 4 �C inmicrotubes. Prior to the experiments, isolates were cultured in po-tato-dextrose-agar (PDA) media for 7 days.

Gallic acid Cafeyc acid Salicylic acid Trans-cinnamic ácid

Fig. 1. Chemical structures of the phenolic acids studied in the present work.

2.4. Phenolic acids treatments

Solutions containing amounts of phenolic acids proportionallyto those found in the Spirulina extract were prepared to assessthe antifungal activity. The concentration of gallic, caffeic acid, sal-icylic acid and trans-cinnamic acids were: 52.2, 33.9, 3.62 and0.30 lg ml�1.

The antifungal activity was tested using a PDA amended withthe phenolic acid solution in a concentration of 3% (w/v), the onewhich reduced 50% of mycelial growth in PDA media comparedto the control, determined previously [14]. Seven treatments,including a control, were tested: LEB-18 extract, gallic acid, caffeicacid, salicylic acid, trans-cinnamic acids, and a mixture of the fouracids (containing 52.2 lg ml�1 gallic, 33.9 lg ml�1 caffeic acid,3.62 lg ml�1 salicylic acid and 0.30 lg ml�1). The aqueous pheno-lic extract was added to the medium at 35–40 �C and afterwardspoured into Petri dishes (10 cm of diameter). Individual acids andthe mixture of four purified acids were prepared with sterile waterand added to the culture medium in the same conditions as theLEB-18 extract.

2.5. Effect on mycelial growth and glucosamine content

Mycelial discs (1.1 cm of diameter) were placed at the center ofeach Petri dish filled with PDA medium. The cultures were incu-bated at 25 �C and colony diameter (cm) was measured daily dur-ing three weeks. The mycelial growth rate (cm day�1) wascalculated at the end of the experiment. The experiment was a fac-torial (4 isolates � 5 phenolic acid treatment) with three replicates.Additionally, a check treatment consisted of fungal growth on PDAwith no phenolic acid. The percent inhibition of the mycelialgrowth (IMG) was calculated, in which: IMG = 100 � ((cm growthin the control � cm growth in the treatment)/cm growth in thecontrol). At day 26, the cultures were frozen and dried at 60 �Cfor 3 h prior to further analysis.

2.6. Glucosamine content

For the quantification of the glucosamine content, each 1 g ofbiomass was dried at 60 �C for 4 h, 5 mL of 6 M HCl was addedand the mixture was sterilized at 100 �C for 40 min. Afterwards,the hydrolysate was neutralized with a 3 M NaOH solution andtitrated with 1% (w/v) KHSO4. The glucosamine content was quan-tified by a colorimetric method using the Erlich reagent (2.67 gDAB-p-dimethylaminobenzaldehyde dissolved in 15 mL of ethanoland 15 mL of hydrochloric acid) and the absorbance was measuredat 530 nm using an analytical curve (0.01–0.2 g L�1) [13,28].

Fig. 2. Chromatogram of the phenolic compounds present in crude extracts of theSpirulina sp. LEB-18 algae strain quantified by reverse phase HPLC-UV.

F.A. Pagnussatt et al. / Pesticide Biochemistry and Physiology 108 (2014) 21–26 23

2.7. Enzime (a-amylase) activity

The enzymatic extract was obtained from the fungal biomasswith 20 mL of 0.9% NaCl in an ultrasonic bath for 40 min, centri-fuged and filtrated. The a-amylase activity was determined bythe degradation of starch quantitatively estimated by the iodomet-ric method [19].

2.8. Mycotoxin extraction and quantification

Mycotoxin extraction followed the QuEChERS method de-scribed in Zachariasova [20]. Briefly, 4 g of a representative samplewere homogenized with 7.5 mL of 0.1% (v/v) formic acid and 10 mLof acetonitrile, and vortexed for 3 min. After the addition of 1 g ofNaCl and 4 g of MgSO4, the mixture was agitated and centrifuged(5 min, 3220g). One mililiter of the supernatant was evaporatedunder nitrogen flux and re-suspended in 1 mL of HPLC-grade ace-tonitrile prior to the chromatographic analysis.

The quantification of nivalenol (NIV), deoxynivalenol (DON) andits acetylates (3-acetyl-DON (3-ADON) and 15-acetyl-DON (15-ADON)) and zearalenone (ZEA) was performed by external stan-dardization. The standards acquired from Sigma Chemicals Cowere diluted in solutions so that they contained 20 lg ml�1 ofthe standard used to construct the analytical curve. The samplewas injected into a HPLC equipped with UV–Vis and fluorescence10AXL detectors and a Discovery� Bio Wide Pore column (C18,25 cm � 4.6 mm, 10 lm, Supelco). The wavelength set in the UVdetector was 219 nm for NIV, DON, 15-Ac-Don and 3-Ac-Don.The wavelengths set in the fluorescence detector were 270 and455 nm, respectively, for emission and excitation of ZEA. The mo-bile phase was water (A): methanol (B) in gradient mode. The gra-dient began at 88% A and 12% B during 8 min. From 9 to 18 min, Bconcentration raised to 100%. At 19 min, the gradient returned to88% A and 12% B and remained in this condition until 27 min.The flow rate was 1 mL min�1 and the injection volume was20 lL. The accuracy of the method was expressed as the percent-age recovery and evaluated by the coefficient of variation of threerepetitions. The limits of detection and quantification were deter-mined by the dilution of standards and fortified samples that gen-erated a detector signal twice as high as the average standarddeviation of the relative area in the retention time of the toxins[21].

2.9. Statistical analysis

The significance of the treatment effects were tested with anal-ysis of variance model and the mean levels of the variables ana-lyzed (glucosamine, a-amylase activity and mycotoxins)whenever significant for the single or interaction effects were com-pared using a Tukey test at 5% significance.

3. Results and discussion

3.1. Phenolic compounds in Spirulina LEB-18 extract

The amount of phenolic compounds found in Spirulina sp. LEB-18 extracts used in this study was 700 lg g�1, using spectrophoto-metric method. The following acids were found in a HPLC: gallic(728 lg g�1), caffeic (42 lg g�1), salicylic (<LQ) and trans-cinnamicacid (<LQ). Previous research identified those four acids and thechlorogenic acid in LEB-18 culture extracts [13,22,23]. Such differ-ence may be related to varying culturing conditions.

The chromatogram of the LEB-18 extract (Fig. 2) depicts the to-tal amount of phenolic acids higher to the value found in the spec-trophotometric quantification, totaling about 110% of the total

phenolic content. This may be attributed to the inherent variabilityof the distinct analytical procedures, which is not consistent. Thecharacterization of the phenolic acids in crude extracts is the firststep to understand the biological activity of each acid and theextract. Several pharmacological activities have already beenattributed to phenolic acids. Gallic acid, for example, showedanti-inflammatory and antimicrobial activity [24], while caffeicacid showed additional antifungal effect [25].

3.2. Inhibition of mycelial growth

For all isolates, mycelial growth curves in the check treatmentand in all single phenolic acids were similar in shape and fungalgrowth reached a similar final diameter of the colony (Fig. 3). Nev-ertheless, in the presence of LEB-18 extract, fungal growth wasgreatly inhibited compared to the other treatments. Radial growthrate of the fungus for the single or mixture of purified phenolicacids and the control treatment ranged from 0.13 to 0.83 cm day�1.For the cultures on LEB-18 extract, radial growth rates ranged from0.01 to 0.45 cm day�1 and the growth curves of all isolates de-picted a potential fungistatic effect during the first nine days ofgrowth (Fig. 3).

For the 07Tr025 strain (F. meridionale), in particular, the addi-tion of standard solutions in the cultures led to mean growth inhi-bitions of 21%, 30%, 33%, 36% and 39% for gallic, caffeic, salicylicand trans-cinnamic, and the mixture of them all, respectively.The LEB-8 extract led to growth inhibitions that ranged between50% and 90% for all isolates. In general, the activity of phenolicacids is related to their chemical structures; based on structuralfeatures of Spirulina phenolic compounds, its leaf extracts could ex-hibit antifungal capacities [26].

3.3. Glucosamine content and amylase activity

In addition to the inhibitory vegetative growth effect, potentialalterations in the cell wall components and target metabolism bythe phenolic acids may suggest their mode of action and thus leadto improved control [27]. In this study, the phenolic acid � isolateinteraction was significant (p < 0.001) for both the glucosaminecontent and the amylase activity. In general, the two F. meridionaleisolates showed glucosamine levels on average 50% lower than thetwo F. graminearum isolates in the control treatments. Glucosa-mine levels were at least 50% lower in all phenolic treatments thanthe control for the two F. graminearum isolates, but a few treat-ments showed significant lower levels of glucosamine for the F.meridionale isolates.

In particular, glucosamine content was lowest for 07Cv022 iso-late in the LEB-8 extract treatment, while no effect was found for

Fig. 3. Mycelial growth curves of the Fusarium meridionale 07 Tr025 (A), Fusarium meridionale 07 Cv022 (B), Fusarium graminearum s. str. 07Tr001 (C) and Fusariumgraminearum s. str. 08Tr024 isolates (D). Control ( ), phenolic extract of Spirulina ( ), gallic acid ( ), caffeic acid ( ), salicylic acid ( ), trans-cinnamic acid ( ), mixture ( ).

24 F.A. Pagnussatt et al. / Pesticide Biochemistry and Physiology 108 (2014) 21–26

the single and mixed phenolic acid standards (Table 2). For the08Tr024 isolate, all phenolic acid standards reduced glucosaminein comparison to the control. Lastly, most of the standards didnot reduce the glucosamine content for the F. meridionale07Cv022 and 07Tr025 isolates. These results demonstrate thatmeasurement of cell wall components, such as glucosamine, maybe a good indicator of cell viability for these fungal species [29].Moreover, it is suggested that phenolic acids are able to penetrate

Table 2Mean glucosamine content (mg g�1) and amylase content (lg of hydrolyzed starch min�1 meffect of phenolic acids.

Treat.1 Glucosamine

07Tr001 08Tr024 07Tr025 07Cv022

C 16.0bB 19.1aA 9.2bC 7.6bD

EX 6.8dB 7.6dA 6.7dBC 6.3dC

GA 7.1dB 3.6eD 4.7dC 11.7aA

CA 17.9aA 9.2bB 9.4bB 9.0cB

SA 11.1bA 8.4cC 8.6cC 10.4abB

TA 7.7cB 7.3dC 10.6aB 11.5aA

MIX 2.8eC 8.4cB 0.9eD 10.6abA

CV 2.33 2.45 2.60 1.77

C: control, EX: phenolic extract of Spirulina, GA: gallic acid, CA: caffeic acid, SA: salicylicicamente (p > 0.05) entre colunas e letras maiúsculas entre linhas. Means followed by difamong colunns and capital letters between lines. CV: variation coefficient.

the cell wall and induce physical and/or chemical alterations or af-fect the metabolic pathway associated to glucosamine production[27].

The amylase activity for the isolates was reduced in the LEB-18extract treatment (Table 2). In general, phenolic acids hamper theachievement of the necessary energy to maintain cell viability, pos-sibly due to the lower availability of substrate and consequently,lower energy to produce the cell wall components. Hence, amylase

g of protein�1) in cultures of Fusarium graminearum species complex strains under the

Amylase activity

07Tr001 08Tr024 07Tr025 07Cv022

11.6fD 108.3aA 27.0cB 18.0dC

5.3gC 8.4dB 4.0eC 15.5deA

12.7eC 33.1cA 21.5cB 21.2cB

25.6aD 32.8cB 61.9aA 31.3bC

15.0cC 51.1bA 44.9bB 14.8eC

13.7dC 6.8eD 56.7aA 16.1deB

16.7bC 52.8bB 17.0dC 87.3aA

2.06 0.85 1.05 1.57

acid, TCA: trans-cinnamic acid, MIX: mixture. Letras minúsculas diferem estatist-ferent lowercase letters present relevant difference through the Tukey test (p > 0.05)

Table 5Effect of phenolic acids on the inhibition of ZEA (lg g�1) by the F. meridionale and F.graminearum isolates.

Treatment F. graminearum F. meridionale

07Tr001 08Tr024 07Tr025 07Cv022

Check 0.02 ± 5.9m 0.52 ± 21.8g <LQ 0.35 ± 9.2i

Spirulina extract 2.55 ± 2.9h 0.66 ± 10.3g 1.04 ± 10.2h 0.45 ± 10.0h

Gallic acid 0.57 ± 14.2k 0.40 ± 8.0h 0.44 ± 7.4j 0.45 ± 9.3i

Caffeic acid 0.10 ± 33.3e ND 0.34 ± 2.9j 0.30 ± 3.5j

Salicylic acid 0.77 ± 8.0j <LQ 0.12 ± 11.8k NDTrans-cinnamic

acidND <LQ 2.84 ± 0.4f <LQ

Mixture of acids 0.31 ± 12.9k <LQ 0.22 ± 18.4k <LQ

Mean ± variation coefficient (n = 3). Means followed by different letters presentrelevant difference through the Tukey test (p > 0.05).

F.A. Pagnussatt et al. / Pesticide Biochemistry and Physiology 108 (2014) 21–26 25

activity may also be an indicator of growth inhibition because thefungus produce digestive enzymes capable of hydrolyzing starchfrom the medium and, thus, obtain energy for growth anddevelopment.

However, for three PA treatments: caffeic acid, salicylic acid andtrans-cinnamic, amylolytic activity was not inhibited in the F.meridionale isolates, which could not be explained. These varia-tions suggest that the species or isolates behave differently in thepresence of phenolic acids, which should be further investigated.

Phenolic acids, besides their known antioxidant and radicalscavenging activity, were reported to reduce inflammation, andact as an antispasmodic and therapeutic agent and an inhibitorof reproduction of the human immunodeficiency virus [30]. Theantimicrobial activity of phenolic acids was reported for caffeic,chlorogenic, ferulic and p-coumaric acids [31]. Caffeic acid andchlorogenic acid were also used to inhibit Listeria monocytogenesand Legionella pneumophila [32], while ferulic acid directly inhib-ited Sclerotinia sclerotiorum [33]. Gallic acid obtained from metha-nolic extracts from Terminalia nigrovenulosa bark showedbiological activities against Fusarium solani, therefore suggestingits potential use for the control of diseases of plants [34].

3.4. Mycotoxin production inhibition

The recovery of the mycotoxins was 122 ± 2%; 106 ± 13% and123 ± 17% for nivalenol, deoxynivalenol and its acetylates, and zea-ralenone, respectively. The concentration of all mycotoxins wassignificantly affected by the interaction of treatments and isolates(p < 0.001). The results of the experimental determination of theinhibition of mycotoxin production showed different responsesagainst the presence of the phenolic extract of Spirulina and stan-dard solutions (Tables 3–5). The phenolic extract of Spirulina inhib-ited the production of NIV and DON of all the isolates with anaverage inhibition of 73% (Table 3).

As to the DON acetylates, higher amounts of 15-ADON com-pared to 3-ADON were produced by all isolates in the control treat-ment. Again, the isolates differed in their responses to the phenolic

Table 3Mean concentration (lg g�1) of deoxynivalenol (DON) and nivalenol (NIV) in cultures of F

Treat.1 DON

07Tr001 08Tr024 07Tr025 07Cv022

C 41.08 ± 1.4a 20.53 ± 0.5a 6.58 ± 1.0e 2.72 ± 2.EX 9.40 ± 1.6c 4.37 ± 4.8d 40.93 ± 0.3a 0.37 ± 5.GA 8.69 ± 1.2d ND 0.26 ± 7.7k 0.30 ± 9.CA 5.62 ± 1.8f ND 0.22 ± 9.1k NDSA 6.54 ± 1.1e ND 0.63 ± 1.6i NDTA ND ND 0.34 ± 2.9j NDMIX 8.11 ± 1.9d 10.0 ± 5.3b 1.89 ± 2.4g ND

C: control, EX: phenolic extract of Spirulina, GA: gallic acid, CA: caffeic acid, SA: salic33.9 lg ml�1 caffeic acid, 3.62 lg ml�1 salicylic acid and 0.30 lg ml�1.

Table 4Mean concentration (lg. g�1) of 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivathe effect of phenolic acids.

Treat 3-ADON

07Tr001 08Tr024 07Tr025 07Cv022

C 2.35 ± 4.0h 1.32 ± 12.7f 3.28 ± 3.7e 5.85 ± 1.7d

EX 4.48 ± 2.2f 1.57 ± 6.7f 3.79 ± 1.9e 2.35 ± 3.3e

GA 2.77 ± 5.9g 1.02 ± 6.4f 3.39 ± 2.7e 1.90 ± 3.0f

CA 2.45 ± 3.3h ND 3.19 ± 3.7e 1.04 ± 4.8 f

SA 1.42 ± 4.4i ND 2.82 ± 1.9f NDTCA ND ND 1.08 ± 5.9h 1.64 ± 4.5f

MIX 1.42 ± 7.4i 1.29 ± 8.3f 4.94 ± 2.0d 1.63 ± 2.9f

C: control, EX: phenolic extract of Spirulina, GA: gallic acid, CA: caffeic acid, SA: salicylic

acid treatments and larger reductions in their amounts were ob-served for the 15-ADON than the 3-ADON mycotoxins. The singlephenolic acids were more effective in reducing the acetylates,especially gallic and caffeic acids, and the salicylic acid andtrans-cinnamic acid did not significantly reduced mycotoxin pro-duction. The main constituents in the biomass of Spirulina weregallic and cafeic acid, with these compounds being the most effec-tive in the inhibition of mycelial growth and mycotoxin productionby Aspergillus flavus (data not published).

Zearalenone production by the isolates was not affected by thephenolic treatments (Table 5) and it is hypothesized that ZEA maybe produced more efficiently by the microorganisms under stressconditions because no ZEA was detected in the control treatment(no phenolic acid). Larger amount of ZEA was found in the presenceof the phenolic extract and gallic acid.

A previous study evidenced the antifungal activity of phenolicacids [14], but mechanisms leading to suppression of mycotoxinproduction are yet to be better understood. The gallic and caffeicacids alone reduced mycotoxin levels produced by the toxigenicspecies, which suggests the need to study the synergistic effect be-tween these two compounds. Apparently, these compounds act asfungal stressors when they hamper the energy obtention due to

usarium graminearum species complex strains under the effect of phenolic acids.

NIV

07Tr001 08Tr024 07Tr025 07Cv022

1e 43.64 ± 4.4a 6.42 ± 3.7c 4.30 ± 1.1d 5.31 ± 3.1d

7i 19.37 ± 1.1b 1.15 ± 7.2f 0.19 ± 16k 2.59 ± 1.0e

3j 0.75 ± 1.3j ND 5.41 ± 1.8c 2.20 ± 4.8e

2.77 ± 2.4h ND 4.55 ± 2.4d 0.53 ± 1.8g

4.50 ± 2.7f ND 1.16 ± 0.9h NDND ND <LQ <LQ

3.18 ± 2.0g 5.60 ± 1.8c 4.47 ± 0.4d <LQ

ylic acid, TCA: trans-cinnamic acid, MIX: mixture containing 52.2 lg ml�1 gallic,

lenol (15-ADON) in cultures of Fusarium graminearum species complex strains under

15-ADON

07Tr001 08Tr024 07Tr025 07Cv022

7.65 ± 1.4e 3.57 ± 4.4d 14.46 ± 4.4b 6.90 ± 1.5c

3.02 ± 3.3g 2.49 ± 4.6e 6.31 ± 4.6c 10.33 ± 1.5a

2.92 ± 0.7g 2.27 ± 7.0e 2.63 ± 6.9f 2.35 ± 2.2e

1.47 ± 4.8i 2.61 ± 3.9g ND 2.70 ± 2.3g

ND ND ND NDND ND <LQ <LQ

3.18 ± 2.0g 5.60 ± 1.8c 4.47 ± 0.4d <LQ

acid, TCA: trans-cinnamic acid, MIX: mixture.

26 F.A. Pagnussatt et al. / Pesticide Biochemistry and Physiology 108 (2014) 21–26

the lower glucose availability [29]. This may trigger the productionof secondary metabolites to compensate and limit the apparentcompetition by the substrate of the medium. Some authors relatean antioxidant activity of the phenolic compounds to the inhibitionof mycotoxin biosynthesis [35]; however, other properties of thesecompounds need to be identified to better understand their modeof action [36].

The individual effect of the four phenolic acids tested in thepresent study had varied across the isolates, a variability that is of-ten reported in the literature [31] and may be due to differences inexperimental methodologies, medium composition and limitationsdue to the poor solubility of many phenolic compounds. It has beenshown that phenolic compounds may have a narrow selectivespectrum of inhibition [37,38]. For example, chlorogenic acidstrongly inhibited Penicillium expansum, Fusarium oxysporum andMucor piriformis, but slightly stimulated the growth of Botrytis cine-rea [39]. In this study, salycilic acid and trans-cinnamic acid inhib-ited mycelial growth of the F. graminearum complex species, butdid not inhibit glucosamine content and amylase activity, forexample.

4. Conclusion

A crude extract of LEB-18 strain of Spirulina sp., mainly consti-tuted of gallic acid, strongly reduced mycelial growth and boththe extract and a purified standard of gallic acid led to the highestreduction in glucosamine content, amylase activity and trichothe-cene concentration produced by toxigenic species of the F. grami-nearum complex.

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