甘肃农业大学：食品科学与工程学院（文献讲义）Use of the Secreted Proteome of Trametes versicolor for Controlling the Cereal Pathogen Fusarium langsethiae.pdf

Intemational Joumal of Molecular Sciences MDPI Article Use of the Secreted Proteome of Trametes versicolor for Controlling the Cereal Pathogen Fusarium langsethiae Alessia ParroniAgnese Bellabarba Marzia Beccaccioli Marzia Scarpari Massimo Reverberiand Alessandro Infantino2 1 Dipartimento di Biologia Ambientale,Sapienza Universita di Roma,Ple Aldo Moro5,00185 Rome,Italy 2 CREA-DC,via C.G.Bertero 22,00156 Roma,Italy Correspondence:massimo.reverberi@uniromal.it Received:1 August 2019;Accepted:22 August 2019;Published:26 August 2019 updates Abstract:Fusarium langsethiae is amongst the most recently discovered pathogens of small grains cereals.F.langsethiae is the main producer,in Europe,of T2 and HT-toxins in small grain cereals albeit often asymptomatic;this makes its control challenging.The European Union(EU)is pushing hard on the use of biocontrol agents to minimize the use of fungicides and pesticides,which are detrimental to the environment and responsible for serious pollution of the soil and superficial water. In line with EU directives(e.g.,128/2009),here we report the use of protein fractions,purified from the culture filtrate of the basidiomycete Trametes versicolor,for controlling F.langsethiae.T.versicolor,a so-called medicinal mushroom which is applied as a co-adjuvant in oncology and other pathologies as a producer of biological response modifiers.In this study,the exo-proteome of T.versicolor proved highly efficient in inhibiting the growth of F.langsethiae and the biosynthesis of the T2 toxin.Results are promising for its future use as a sustainable product to control F.langsethiae infection in cereals under field conditions. Keywords: Fusarium langsethiae;exo-proteome;Trametes versicolor;T2-HT2;mycotoxins growth inhibition 1.Introduction Fungi are organisms comprising yeasts,molds and mushrooms and they have been used for long time both in medicine as co-therapy and as food for their nutritional value.Notably,traditional Chinese medicine considered mushrooms(in particular basidiomycetes)as a useful source of bioactive compounds of large interest in medicine and to strengthen the welfare of the human body [1]. Polysaccharides,proteins,glycoproteins and peptides from different mushrooms(i.e.,Lentinula edodes, Ganoderma lucidum,Trametes versicolor)demonstrated antibacterial,antiviral,antitumor and immune modulatory activity.One of the most known representatives of these compounds is the lentinan, a cell wall polysaccharide extracted from Lentinula edodes known for its medicinal properties [2-4]. Moreover,recent studies demonstrate a promising preclinical antileukemia activity of Tramesan,a patented a-hetero-polysaccharide purified from culture filtrate of T.versicolor [5].Other biological effects were also investigated on murine cell line of melanoma(B16)where Tramesan showed,due to its reactive oxygen species (ROS)-indirect-scavenging ability,a significant limitation of cell growth. Tramesan,in fact,increased melanin content enhancing nf-2 expression and protecting melanocytes against the dangerous ROS effect(due to the high intrinsic oxidative stress expressed by cancer cells) and,finally,a significant reduction of cell growth [6].Another group of molecules with medicinal properties,such as proteins and peptides from higher basidiomycetes,has attracted the interest of Imt.1Mol.Sci.2019,20,4167;dot10.3390/ims20174167 www.mdpi.com/journal/ijms

International Journal of Molecular Sciences Article Use of the Secreted Proteome of Trametes versicolor for Controlling the Cereal Pathogen Fusarium langsethiae Alessia Parroni 1 , Agnese Bellabarba 1 , Marzia Beccaccioli 1 , Marzia Scarpari 2 , Massimo Reverberi 1,* and Alessandro Infantino 2 1 Dipartimento di Biologia Ambientale, Sapienza Università di Roma, P.le Aldo Moro 5, 00185 Rome, Italy 2 CREA-DC, via C.G. Bertero 22, 00156 Roma, Italy * Correspondence: massimo.reverberi@uniroma1.it Received: 1 August 2019; Accepted: 22 August 2019; Published: 26 August 2019 Abstract: Fusarium langsethiae is amongst the most recently discovered pathogens of small grains cereals. F. langsethiae is the main producer, in Europe, of T2 and HT-toxins in small grain cereals, albeit often asymptomatic; this makes its control challenging. The European Union (EU) is pushing hard on the use of biocontrol agents to minimize the use of fungicides and pesticides, which are detrimental to the environment and responsible for serious pollution of the soil and superficial water. In line with EU directives (e.g., 128/2009), here we report the use of protein fractions, purified from the culture filtrate of the basidiomycete Trametes versicolor, for controlling F. langsethiae. T. versicolor, a so-called medicinal mushroom which is applied as a co-adjuvant in oncology and other pathologies as a producer of biological response modifiers. In this study, the exo-proteome of T. versicolor proved highly efficient in inhibiting the growth of F. langsethiae and the biosynthesis of the T2 toxin. Results are promising for its future use as a sustainable product to control F. langsethiae infection in cereals under field conditions. Keywords: Fusarium langsethiae; exo-proteome; Trametes versicolor; T2-HT2; mycotoxins; growth inhibition 1. Introduction Fungi are organisms comprising yeasts, molds and mushrooms and they have been used for long time both in medicine as co-therapy and as food for their nutritional value. Notably, traditional Chinese medicine considered mushrooms (in particular basidiomycetes) as a useful source of bioactive compounds of large interest in medicine and to strengthen the welfare of the human body [1]. Polysaccharides, proteins, glycoproteins and peptides from different mushrooms (i.e., Lentinula edodes, Ganoderma lucidum, Trametes versicolor) demonstrated antibacterial, antiviral, antitumor and immune modulatory activity. One of the most known representatives of these compounds is the lentinan, a cell wall polysaccharide extracted from Lentinula edodes known for its medicinal properties [2–4]. Moreover, recent studies demonstrate a promising preclinical antileukemia activity of Tramesan, a patented α-hetero-polysaccharide purified from culture filtrate of T. versicolor [5]. Other biological effects were also investigated on murine cell line of melanoma (B16) where Tramesan showed, due to its reactive oxygen species (ROS)—indirect—scavenging ability, a significant limitation of cell growth. Tramesan, in fact, increased melanin content enhancing nrf-2 expression and protecting melanocytes against the dangerous ROS effect (due to the high intrinsic oxidative stress expressed by cancer cells) and, finally, a significant reduction of cell growth [6]. Another group of molecules with medicinal properties, such as proteins and peptides from higher basidiomycetes, has attracted the interest of Int. J. Mol. Sci. 2019, 20, 4167; doi:10.3390/ijms20174167 www.mdpi.com/journal/ijms

mt.1M6l.sci2019,204167 2of14 the scientific community.In particular proteases,defensins,lectins,laccases,polysaccharopeptides and immune modulatory proteins showed different medicinal properties [1].A particular family of aaaeemya ucidumt and other mu shrooms are denomin small protein molecules with a molecular weight of about 13kD with immune regulating activity [7 uously increasing. tools while fo y lant d Plant patho erapeutic organisms and metabolites causing se omic losses in ag indeed.mycotoxins ar dangerous for animals and humans,often showing toxic or carcinogenic effects towards various target organs (i.e.aflatoxins vs.liver,ochratoxins vs.kidney,trichothecenes vs.lymphoid organs).In previous studies,we focused our attention on non-toxic or edible basidiomycetes such as Lentimla edodes and T.versicolor.Polysaccharide fraction purified from culture filtrates of L edodes showed a significant inhibitin stra ially m 1,121.Ex bot san was af active in the ntrol of the infection of wheat l by strengthening the defenses of plant against the fun was non-toxic for fungal pathogens,whereas using the whole filtrate (i.e.without any purification step)showed an interesting fungitoxic effect(M.Reverberi,personal communication).The promising with these studies prompted us to investigate the protein fractions( the total filtrate d.w.)of T.versicolor as source of bioactive compounds and their possible efficacy ir e pathoge 1 to ostudy the exo-proteor Prot ie lac nd ymes the Fusarium langsethiae is an ascomycete located in the Gibberella-clade of Fusarium,in which several pathogens of small grains cereals(such as oat,wheat and barley)are present,which are very detrimental for crop quality and safety [14].These pathogens are widespread worldwide and are actually producers some of which are even regulated by C to mit their dangerous eneson imals [15,16].Fu among th W S ed an cer in the T2 nd HT2 toxins (type hute and the detectio of the fun al infoction has to he ir nted with molecular echniques.in italy.the pathogen incidence is higher in centre and southern than in northern regions being related to climate conditions characterized by high temperature and scarce rain fallings during the wheat flowering [16].The toxins T2 and HT2 produced by F.langsethine present high toxicity probably due to their lipophilicity and are consequently likely to penetrate the cells[19].Their toxic effects regard the inhil and RN nd pr eins ar ction of lymphocytes ofthe cell in s ted [201 ney ca TA The would represent an important goal to be achieved for the safe and security of foods and feeds.Scarce literature is available on the fight against F.langsethine.This is probably due to the difficulty to detect pathogenicity symptoms.At the same time,scarce reports are available about bioagents efficient against F.langsethine.These reasons prompted us to investigate bioagents from T.versicolor to control this

Int. J. Mol. Sci. 2019, 20, 4167 2 of 14 the scientific community. In particular proteases, defensins, lectins, laccases, polysaccharopeptides and immune modulatory proteins showed different medicinal properties [1]. A particular family of immune modulatory proteins similar to phytohemagglutinins and immunoglobulins from Ganoderma lucidum and other mushrooms are denominated FIP (fungal immunomodulatory proteins). They are small protein molecules with a molecular weight of about 13kD with immune regulating activity [7] and the number of proteins belonging to FIP family is continuously increasing. Several studies, however, mainly deal with the bioactivity of mushroom compounds as therapeutic tools while few studies regard the control of plant diseases. Plant pathogens and mycotoxins are organisms and metabolites causing severe economic losses in agriculture; indeed, mycotoxins are dangerous for animals and humans, often showing toxic or carcinogenic effects towards various target organs (i.e., aflatoxins vs. liver, ochratoxins vs. kidney, trichothecenes vs. lymphoid organs). In previous studies, we focused our attention on non-toxic or edible basidiomycetes such as Lentinula edodes and T. versicolor. Polysaccharide fraction purified from culture filtrates of L. edodes showed a significant inhibiting effect on aflatoxin synthesis by the plant pathogens Aspergillus flavus and A. parasiticus [8–10] and the same inhibiting effect on aflatoxin synthesis was demonstrated by rough and partially purified extracts from T. versicolor [11,12]. Extracts from both mushrooms promoted antioxidant defenses of the fungal cells consequently inhibiting toxin synthesis. In other studies, Tramesan was effective in the control of the infection of wheat leaves by the pathogen Parastagonospora nodorum [6] by strengthening the defenses of plant against the fungal pathogen. Nevertheless, Tramesan was non-toxic for fungal pathogens, whereas using the whole filtrate (i.e., without any purification step) showed an interesting fungitoxic effect (M. Reverberi, personal communication). The promising results obtained with these studies prompted us to investigate the protein fractions (up to 15% of the total filtrate d.w.) of T. versicolor as source of bioactive compounds and their possible efficacy in controlling other fungal pathogens and toxins synthesis. The idea to study the exo-proteome was also reinforced by the biological role played by FIP proteins from mushrooms. Previous research, in fact, evidenced a significant efficacy of oxidase enzymes (i.e., laccases), from the culture filtrates of an isolate of T. versicolor, in the detoxification and degradation of different toxins such as aflatoxins, ochratoxin A, Fusarium toxins (Deoxynivalenol and Fumonisin B1) at different inhibiting levels [13]. Fusarium langsethiae is an ascomycete located in the Gibberella-clade of Fusarium, in which several pathogens of small grains cereals (such as oat, wheat and barley) are present, which are very detrimental for crop quality and safety [14]. These pathogens are widespread worldwide and are actually producers of different toxins; some of which are even regulated by EC to limit their dangerous effects on humans and animals [15,16]. Fusarium langsethiae can be included among the new species discovered and studied over recent years, being the main producer in Europe of the T2 and HT2 toxins (type A trichothecenes) [15,17] found on small grain cereals [18]. It is difficult to investigate the pathogenicity of this fungus due to lack of visible symptoms on the infected plants. For this behavior, it is considered as an endophyte and the detection of the fungal infection has to be implemented with molecular techniques. In Italy, the pathogen incidence is higher in Centre and Southern than in Northern regions being related to climate conditions characterized by high temperature and scarce rain fallings during the wheat flowering [16]. The toxins T2 and HT2 produced by F. langsethiae present high toxicity probably due to their lipophilicity and are consequently likely to penetrate the cells [19]. Their toxic effects regard the inhibition of synthesis of DNA and RNA and proteins and the reduction of lymphocytes and immune defenses [15]. Moreover, they can induce lipid peroxidation affecting the membrane functions of the cell infected [20]. T2 toxin causes the ATA (Alimentary Toxic Aleukia) in humans. Therefore, contamination of small cereals such as wheat with F. langsethiae may represent a serious concern for human and animal health. Reducing the incidence of F. langsethiae in small grain cereals would represent an important goal to be achieved for the safe and security of foods and feeds. Scarce literature is available on the fight against F. langsethiae. This is probably due to the difficulty to detect pathogenicity symptoms. At the same time, scarce reports are available about bioagents efficient against F. langsethiae. These reasons prompted us to investigate bioagents from T. versicolor to control this

Int. J. Mol. Sci. 2019, 20, 4167 3 of 14 pathogen. In this study, we report the ability of different fractions of Trametes versicolor exo-proteome to inhibit F. langsethiae growth and toxin synthesis; we here propose these fractions as a possible biocontrol tool for managing this pathogen and its toxins. 2. Results 2.1. Culture Filtrate (CF) of Trametes Versicolor Inhibits the Growth of Fusarium Langsethiae The preliminary assays concerned the effect of cultural filtrate (CF) of T. versicolor added to PDA medium in Petri dishes at concentrations of 0.04% w/v and 0.08% w/v on the growth of the pathogen F. langsethiae incubated at 25 ◦C for 3, 5 and 7 days after incubation (dai) (Figure S1). The growth of the pathogen resulted in the inhibition of 53.8% at the concentration of 0.04% w/v and 61.4% at the concentration of 0.08% w/v after 5 dai in comparison with the untreated sample (Figure 1). At 7 dai, however, the pathogen growth slightly increased even if significant inhibition was maintained in comparison with the control (t test; p < 0.001 for both treatments versus control). The fungal growth was estimated by diameter growth. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 15 exo-proteome to inhibit F. langsethiae growth and toxin synthesis; we here propose these fractions as a possible biocontrol tool for managing this pathogen and its toxins. 2. Results 2.1. Culture Filtrate (CF) of Trametes Versicolor Inhibits the Growth of Fusarium Langsethiae The preliminary assays concerned the effect of cultural filtrate (CF) of T. versicolor added to PDA medium in Petri dishes at concentrations of 0.04% w/v and 0.08% w/v on the growth of the pathogen F. langsethiae incubated at 25 °C for 3, 5 and 7 days after incubation (dai) (Figure S1). The growth of the pathogen resulted in the inhibition of 53.8% at the concentration of 0.04% w/v and 61.4% at the concentration of 0.08% w/v after 5 dai in comparison with the untreated sample (Figure 1). At 7 dai, however, the pathogen growth slightly increased even if significant inhibition was maintained in comparison with the control (T test; p < 0.001 for both treatments versus control). The fungal growth was estimated by diameter growth. Figure 1. Growth inhibition of F. langsethiae (1595) treated with 0.04 and 0.08 % w/v of CF TV117. Histograms of the growth during the time (3, 5 and 7 dai) and the percentage of the growth inhibition in comparison with the control (ctr) (100%) is reported. The data are the mean ± SD of 3 experiments. 2.2. SDS-PAGE of the Exo-Proteome of Trametes Versicolor The encouraging results obtained on the pathogen growth inhibition pushed us to characterize the protein fraction of the CF TV117. In fact, we already know that the polysaccharide fraction of the CF did not show fungal growth inhibition [6,9,11]. To this aim, proteins present in CF TV117 were precipitated with ammonium sulfate (AS) at 75 and 90% concentrations. The 2 protein fractions (F90AS and F75AS) obtained were analyzed by SDS-PAGE (Figure 2). In general, F90AS presented a lower type and quantity of proteins in comparison with F75AS fraction. This was confirmed by Bradford assay (data not shown). The molecular weights of the fractions assayed are in the range of 40–75 kDa in the F90AS fraction—the higher quantity of proteins localized at about 50 kDa—whereas the proteins in F75AS were distributed in a wider range—from 15 to 100 kDa (Figure 2). Figure 1. Growth inhibition of F. langsethiae (1595) treated with 0.04 and 0.08% w/v of CF TV117. Histograms of the growth during the time (3, 5 and 7 dai) and the percentage of the growth inhibition in comparison with the control (ctr) (100%) is reported. The data are the mean ± SD of 3 experiments. 2.2. SDS-PAGE of the Exo-Proteome of Trametes Versicolor The encouraging results obtained on the pathogen growth inhibition pushed us to characterize the protein fraction of the CF TV117. In fact, we already know that the polysaccharide fraction of the CF did not show fungal growth inhibition [6,9,11]. To this aim, proteins present in CF TV117 were precipitated with ammonium sulfate (AS) at 75 and 90% concentrations. The 2 protein fractions (F90AS and F75AS) obtained were analyzed by SDS-PAGE (Figure 2). In general, F90AS presented a lower type and quantity of proteins in comparison with F75AS fraction. This was confirmed by Bradford assay (data not shown). The molecular weights of the fractions assayed are in the range of 40–75 kDa in the F90AS fraction—the higher quantity of proteins localized at about 50 kDa—whereas the proteins in F75AS were distributed in a wider range—from 15 to 100 kDa (Figure 2)

Int. J. Mol. Sci. 2019, 20, 4167 4 of 14 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 of 15 Figure 2. SDS-PAGE of F75AS (left line) and F90AS (right line) protein fractions. Std is the protein marker (middle line). The molecular range of std is from 11 to 245 kDa. 2.3. Bioactivity Assay of F75AS and F90AS Fractions on F. Langsethiae Growth Biological assay was performed to verify whether the different fractions (F75AS and F90AS, 80 μg each) had an inhibiting effect on F. langsethiae growth. The assay was performed on a Biolog FF Microplate with 96 wells, as reported in Materials and Methods. F75AS and F90AS fractions inhibited fungal growth at different level (Figure 3). F90AS fraction inhibited fungal growth both at 4 and 7 dai more than the F75AS fraction. In fact, the results showed that F90AS was more efficient than F75: 71.1% versus 11.1% at 4 dai (T test, p < 0.001 and p = 0.06, respectively) and 82.4 versus 46.4 at 7 dai, respectively (T test, p < 0.001 for both treatments). The effect of both fractions on the fungal growth during the time was unchanged. Figure 2. SDS-PAGE of F75AS (left line) and F90AS (right line) protein fractions. Std is the protein marker (middle line). The molecular range of std is from 11 to 245 kDa. 2.3. Bioactivity Assay of F75AS and F90AS Fractions on F. Langsethiae Growth Biological assay was performed to verify whether the different fractions (F75AS and F90AS, 80 µg each) had an inhibiting effect on F. langsethiae growth. The assay was performed on a Biolog FF Microplate with 96 wells, as reported in Materials and Methods. F75AS and F90AS fractions inhibited fungal growth at different level (Figure 3). F90AS fraction inhibited fungal growth both at 4 and 7 dai more than the F75AS fraction. In fact, the results showed that F90AS was more efficient than F75: 71.1% versus 11.1% at 4 dai (t test, p < 0.001 and p = 0.06, respectively) and 82.4 versus 46.4 at 7 dai, respectively (t test, p < 0.001 for both treatments). Int. J. Mol. Sci. The effect of both fractions on the fungal growth during the time was unchanged. 2019, 20, x FOR PEER REVIEW 5 of 15 Figure 3. Fungal growth (absorbance at 750 nm) assayed by Biolog FF microplate, after 4 and 7 dai at 25 °C in presence (F75 AS and F90 AS) and in the absence (ctr) of protein fractions. The results are the mean ± SD of 3 different experiments. 2.4. Further Fractionation of F75AS and F90AS by Sephacryl S-100 The protein fractions F75AS and F90AS were subsequently fractionated by size exclusion chromatography (SEC), using Sephacryl S-100 column (Figure S2 A and B). The chromatograms obtained from F75AS (Figure S2A) and F90AS (Figure S2B) allowed to individuate protein subfractions numbered as F75 (1–8) and F90 (1–6) based on their retention time and molecular weight (MW). As evidenced, in F75-SEC (Figure S2A) different peaks were showed; this indicated that proteins were fractionated within a wide range of MW. The F90-SEC (Figure S2A) shaped more like a Gaussian curve indicating a narrower MW range of the proteins. 2.5. Bioactivity of the Sub-Fractions F75 (1–8) and F90 (1–6) on F. Langsethiae Growth and Mycotoxin Production The bioactivity of the sub-fractions obtained from F75AS and F90AS was analyzed in the same experimental conditions of the previous assays. After 7 dai at 25 °C the protein fractions F75_7 and F90_2, 4, 5 markedly inhibited fungal growth (Figure 4 A and B). Concerning the F75 sub-fractions, up to 2 dai of no significant growth inhibition in respect of the control was evidenced. After this time the fractions F75_1, 2, 6, 7, 8 worked better than the others. As regards the F90 sub-fractions, the trend of growth inhibition is similar to the F75 sub-fractions up to 2 dai. After this point, in this case all the fractions, at different level, were able to inhibit the fungal growth. Considering the last time point at which the growth was registered, i.e., 7 dai, the fractions F75_7, F90_2, F90_4 and F90_5 were the most effective in limiting the fungal growth. Under these conditions, the T2 toxin was analyzed at 7 dai. At this time (7 dai), we tested the ability of the different sub-fractions in limiting T-2 toxin biosynthesis by F. langsethiae under the same cultural conditions (Figure 5). F90_4 and F90_5 inhibited about 60% and F75_7 and F90_2 about 98% T2 biosynthesis. Concerning the relationship between T2 production and fungal growth, as shown in the table 1 below, the fungal growth resulted similar in F75_7, F90_2, 4, 5 fractions, in comparison with the control but the T2 toxin production was 2% in F75_7 and F90_2 and 40% in the other, respect to T2 production in the control. Figure 3. Fungal growth (absorbance at 750 nm) assayed by Biolog FF microplate, after 4 and 7 dai at 25 ◦C in presence (F75 AS and F90 AS) and in the absence (ctr) of protein fractions. The results are the mean ± SD of 3 different experiments

Int. J. Mol. Sci. 2019, 20, 4167 5 of 14 2.4. Further Fractionation of F75AS and F90AS by Sephacryl S-100 The protein fractions F75AS and F90AS were subsequently fractionated by size exclusion chromatography (SEC), using Sephacryl S-100 column (Figure S2A,B). The chromatograms obtained from F75AS (Figure S2A) and F90AS (Figure S2B) allowed to individuate protein sub-fractions numbered as F75 (1–8) and F90 (1–6) based on their retention time and molecular weight (MW). As evidenced, in F75-SEC (Figure S2A) different peaks were showed; this indicated that proteins were fractionated within a wide range of MW. The F90-SEC (Figure S2A) shaped more like a Gaussian curve indicating a narrower MW range of the proteins. 2.5. Bioactivity of the Sub-Fractions F75 (1–8) and F90 (1–6) on F. Langsethiae Growth and Mycotoxin Production The bioactivity of the sub-fractions obtained from F75AS and F90AS was analyzed in the same experimental conditions of the previous assays. After 7 dai at 25 ◦C the protein fractions F75_7 and F90_2, 4, 5 markedly inhibited fungal growth (Figure 4A,B). Concerning the F75 sub-fractions, up to 2 dai of no significant growth inhibition in respect of the control was evidenced. After this time the fractions F75_1, 2, 6, 7, 8 worked better than the others. As regards the F90 sub-fractions, the trend of growth inhibition is similar to the F75 sub-fractions up to 2 dai. After this point, in this case all the fractions, at different level, were able to inhibit the fungal growth. Considering the last time point at which the growth was registered, i.e., 7 dai, the fractions F75_7, F90_2, F90_4 and F90_5 were the most effective in limiting the fungal growth. Under these conditions, the T2 toxin was analyzed at 7 dai. At this time (7 dai), we tested the ability of the different sub-fractions in limiting T-2 toxin biosynthesis by F. langsethiae under the same cultural conditions (Figure 5). F90_4 and F90_5 inhibited about 60% and F75_7 and F90_2 about 98% T2 biosynthesis. Concerning the relationship between T2 production and fungal growth, as shown in the Table 1 below, the fungal growth resulted similar in F75_7, F90_2, 4, 5 fractions, in comparison with the control but the T2 toxin production was 2% in F75_7 and F90_2 and 40% in the other, respect to T2 production in the control. Table 1. Fungal growth and T2 toxin production by F. langsethiae non treated (ctr) and treated with protein fractions F75_7, F90_2, F90_4, F90_5 at 7 dai at 25 ◦C. Fractions Growth (%) T2 Toxin (%) ctr 100 100 F75_7 12.5 2 F90_2 11.7 2 F90_4 12.5 40 F90_5 12.5 40