Preliminary Study on the Use of Chitosan as an Eco-Friendly Alternative to Control Fusarium Growth and Mycotoxin Production on Maize and Wheat

The objectives of the present study were to determine the combined effects of chitosan and water activity (aW) on growth and mycotoxin production in situ on the two most important Fusarium species (F. proliferatum and F. verticillioides) present on maize, and on F. graminearum, the main pathogen causing Fusarium head blight on wheat. Results showed that low-molecular-weight chitosan with more than 70% deacetylation at the lowest dose used (0.5 mg/g) was able to reduce deoxynivalenol (DON) and fumonisin (FBs) production on irradiated maize and wheat grains. Growth rates of F. graminearum also decreased at the lowest chitosan dose used (0.5 mg/g), while F. verticillioides and F. proliferatum growth rates were reduced at 0.98 aW at the highest chitosan dose used (2 mg/g). Since mycotoxins are unavoidable contaminants in food and feed chains, their presence needs to be reduced in order to minimize their effects on human and animal health and to diminish the annual market loss through rejected maize and wheat; in this scenario, pre- and post-harvest use of chitosan could be an important alternative.


Introduction
Fusarium is one of the most economically important genera of phytopathogenic fungi. Several Fusarium species can infect small grain cereals (wheat, barley, and oat) and maize causing losses by seedling blight or reducing seed germination, or by causing seedling foot and stalk rots. However, the most important diseases in cereals, due to a severe reduction in yield and quality, are head blight of small cereals such as wheat, and ear rot of maize. Also, another risk is the presence of Fusarium toxins that contaminate cereals and are of concern because they could result in harmful contamination of foods and feedstuffs [1]. Maize can be infected by many toxigenic fungi [1][2][3], including Fusarium verticillioides and F. proliferatum [3][4][5][6][7]. These Fusarium species belong to the Fusarium fujikuroi species complex (FFSC) and are important pathogens of maize that can produce a high number of mycotoxins. Among them, fumonisins (FBs) are the most important in terms of occurrence and levels. Fumonisins are polyketides which are structurally similar to cellular sphingolipids, and they were shown to inhibit sphingolipid biosynthesis via the ceramide synthase pathway. Fumonisin toxicity is thought to result from the blockage of sphingolipid biosynthesis [8]. Dietary exposure to these mycotoxins was associated with leukoencephalomalacia in equine, hepatic, and renal toxicity in rodents, pulmonary In a previous study in vitro, we demonstrated the effect of chitosan on growth of F. verticillioides and F. proliferatum strains, as well as on FB production, under different a W at 25 • C, using a maize-based media. We observed that chitosan was able to significantly reduce growth rate and FB production, with maximum levels of reduction in both parameters obtained at the highest doses used [42]. Ramirez et al. [30] suggested that the effect of antifungal compounds on artificial substrate may not accurately represent the real situation on a natural substrate. The use of irradiated grains that retained viability may be a more appropriate system for screening antifungal and anti-mycotoxin compounds.
Under this scenario, chitosan could be proposed as a possible option to control fungal growth and mycotoxin accumulation in cereals, mainly due to its biological properties and its easy production by partial alkaline N-deacetylation of chitin [43].
The objectives of the present study were to determine the effect of chitosan under different a W at 25 • C on the (i) lag phase, (ii) growth rate, and (iii) mycotoxin (fumonisin and dexynivalenol) production by F. verticillioides, F. proliferatum, and F. graminearum on irradiated maize and wheat grains.

Chitosan Characterization
The average chitosan molecular weight (Mw) in g/mol determined by the Mark-Houwink-Sakurada equation was 3.42 ± 0.08 × 10 3 g/mol. The deacetylation degree (DD), determined by an infrared spectroscopy analysis, was 77.6%. Figure 1 summarizes the effect of a W and chitosan doses on F. proliferatum RC2080 and F. verticillioides M7075 growth rates on irradiated maize grains. The results showed that increasing doses of chitosan at 0.99 a W in both Fusarium species did not affect their growth rates, while at 0.98 a W and 2 mg/mL chitosan, growth rates were significantly lower for both Fusarium species. Significant increases (p > 0.001) in growth rates were observed when F. proliferatum RC2080 and F. verticillioides M7075 were treated with chitosan at 0.95 a W ; the increases were around 125% and 181% for F. proliferatum RC2080 and F. verticillioides M7075, respectively. No statistical differences were observed (p > 0.001) when F. proliferatum RC2080 was treated with chitosan doses of 0.5 and 1 mg/mL and 0.99 and 0.98 a W , regarding the control treatment. F. verticillioides M7075 treated with chitosan doses of 0.5 and 1 mg/mL and 0.99 a W showed no statistical differences (p > 0.001) compared to the control.

Effect of Chitosan Concentration and a W on Growth Rates
The statistical analysis using ANOVA of single factors (strain, chitosan dose, and a W ) and of all interactions (two and three-way) on growth rate showed that the major effect was due to a W followed by the interaction chitosan × a W dose. The strain factor did not significantly affect the growth rate; therefore, it leads us to conclude that the strain behavior was the same in every condition tested (Table 1).    Figure 2 summarizes the effect of a W and chitosan doses on F. graminearum strain growth rates on irradiated wheat grains. Under control conditions, maximum growth rates were observed at 0.995 and 0.98 a W depending on the F. graminearum strain tested. Growth rates of both F. graminearum strains significantly decreased when chitosan was used, as compared to the control conditions ( Figure 2). The highest growth reduction with chitosan was observed at 0.98 a W for both strains. However, no significant differences were observed among chitosan concentrations at the same a W for both strains.
Pathogens 2018, 7, x FOR PEER REVIEW 5 of 15 Figure 2 summarizes the effect of aW and chitosan doses on F. graminearum strain growth rates on irradiated wheat grains. Under control conditions, maximum growth rates were observed at 0.995 and 0.98 aW depending on the F. graminearum strain tested. Growth rates of both F. graminearum strains significantly decreased when chitosan was used, as compared to the control conditions ( Figure  2). The highest growth reduction with chitosan was observed at 0.98 aW for both strains. However, no significant differences were observed among chitosan concentrations at the same aW for both strains.
The statistical analysis using ANOVA of single factors (strains, aW, and chitosan dose) and their interactions on growth rates showed that aW and chitosan dose alone, as well as chitosan × aW interaction, were significant (p < 0.001), whereby chitosan was the factor that had the greatest effect on growth rates of both F. graminearum strains ( Table 2).  The statistical analysis using ANOVA of single factors (strains, a W , and chitosan dose) and their interactions on growth rates showed that a W and chitosan dose alone, as well as chitosan × a W interaction, were significant (p < 0.001), whereby chitosan was the factor that had the greatest effect on growth rates of both F. graminearum strains ( Table 2).

Effect of Chitosan Concentration and a W on Mycotoxin Production
The F. verticillioides strain was able to produce higher amounts of fumonisins in almost all tested conditions in comparison with F. proliferatum. Maximum fumonisin production in the control condition for both strains was observed at 0.98 a W , and, at that a W , a significant reduction in FB levels was observed when chitosan treatments were used, reaching 88% and 95% reduction for F. proliferatum RC2080 and F. verticillioides M7075, respectively. For both strains, the lowest amounts of FBs were produced at 0.95 a W in the control condition. A high stimulation in fumonisin production by F. verticillioides M7075 was observed when the chitosan dose was 1 mg/g at 0.99 a W (Table 3); for the same strain, no significant differences were observed at 0.95 a W when chitosan was applied, while, for F. proliferatum, when chitosan dose was ≥1 mg/g at the same a W , FB production was significantly reduced. For both strains, in all conditions, FB 1 was found in higher amounts than FB 2 and FB 3 (data not shown). The statistical analysis using ANOVA showed that single factors (strains, a W , and chitosan dose) and some interactions (S × a W and C × a W ) were significant on fumonisin production. The major effect was given by a W , followed by strain factor (Table 4).  Regarding DON production, maximum concentrations were produced by the two F. graminearum strains at 0.995 a W in the control condition (without chitosan addition) on irradiated wheat grains. When chitosan treatments were applied to the irradiated wheat grains, DON levels were lower in comparison with the control condition, with the exception of F. graminearum RC22-2 at 0.99 a W , where a stimulation in DON production was observed. Overall, it was observed that the F. graminearum RC22-2 strain produced higher amounts of DON in comparison with the F. graminearum RCFG6001 strain (Table 5). DON production by F. graminearum RCFG6001 strain was reduced as long as increasing chitosan doses were used, with reductions between 93% and 100% in all treatments and a W tested. DON production by F. graminearum RC22-2 was reduced at 0.995 and 0.98 a W when chitosan was applied at all used doses, with the exception of 0.98 a W when 1 mg/g was used and a slight stimulation in DON production was observed. Nevertheless, it was not significant ( Table 5).
The analysis of the effect of individual factors (strains, chitosan dose, and a W ) and most interactions (two-and three-way) on DON production showed that all the factors and interactions were statistically significant. Strain factor was the variable that most affected DON production, followed by chitosan dose and a W (Table 4).

Discussion
The present study highlights the potential of chitosan for controlling both growth and mycotoxin production (FBs and DON) by important Fusarium species, which are common cereal contaminants. However, it is important to consider that different responses to chitosan use were found among different fungal strains. Notably, at the lowest chitosan concentration used, we observed reductions in growth rates and an important effect on DON production by both F. graminearum strains, independently of the a W . The only exception was observed at 0.99 a W for strain RC22-2.
Regarding the Fusarium species that produce fumonisins (F. verticillioides and F. prolifeartum), we only observed a significant reduction in growth rates at 0.98 aw for both species at the highest chitosan dose used. Overall, important FB reductions were found at the lowest doses used (0.5 mg/g) at 0.98 a W , which is considered the most suitable a W for this mycotoxin production on maize [44]. Moreover, for the other a W , the most effective chitosan dose for fumonisin reduction was 1 mg/g with the exception of F. verticillioides at 0.99 a W .
The antifungal activity of chitosan over a broad range of phytopathgenic fungi was the subject of several review papers [35,[45][46][47]. To the best of our knowledge, none of the previous studies reported on the interactions between the efficacy of chitosan or chitosan derivates and a W or temperature, which were demonstrated to be key parameters determining germination, growth, and mycotoxin production by different fungal species [20,48].
Also, there are no other studies in the literature evaluating both the chitosan and a W effect on Fusarium species growth and mycotoxin production on irradiated grains. There was only one study in vitro previously carried out in our laboratory by Ferrochio et al. [42], who demonstrated that different chitosan doses from 0.5 to 3.0 mg/L, combined with a W reduction, increased lag phases and decreased mycelial growth rates in F. verticillioides and F. proliferatum strains, when the study was performed in a wheat-based media at 25 • C. The lowest growth rate was obtained at the highest chitosan concentration tested (3 mg/mL) and at the lowest a W (0.93). However, these results are different from those obtained in the present study using the same F. verticillioides and F. proliferatum strains on irradiated maize grains. This could be explained by the fact that, although irradiated grains contained all the nutritional elements in order to allow the fungal development, they could represent a natural barrier in the penetration of Fusarium, thus restricting the fungal growth. However, good levels of fumonisin reduction were achieved in both studies at the lowest chitosan doses used (0.5 mg/g).
The antifungal activities of chitosan relied on several intrinsic and extrinsic factors, such as pH, fungal species, presence or absence of metal cations, pKa, Mw, DD, etc. [35]. Verlee et al. [45] criticized that most papers dealing with antifungal activity did not report the characterization (Mw and DD) of chitosan. For all these reasons, during the present work, some characteristics of the commercial chitosan used were determined. The results showed that chitosan presents a low molecular weight and 71% DD. For soluble chitosan, pH is a crucial factor related to solubility, and can further alter antifungal activity. The antifungal activity of chitosan is exhibited when the pH is below the representative pKa (~6.5), the value at which the soluble molecule could be dissociated as ions. In the present study, the pH of the chitosan solution was maintained below the pKa.
There were several attempts, in the last few years, to use chitosan for reducing FHB and DON contamination. Khan et al. [49] showed that both chitosan and Pseudomonas fluorescens (biological control agent) were very effective in reducing FHB disease caused by F. culmorum and DON contamination in wheat and barley grains, when they were applied at high disease pressure under glasshouse and field experiments. They concluded that, overall, chitosan was more effective than the potential biocontrol bacterium P. fluorescens strain MKB158. Also, Kheiri et al. [50], reported a good in vitro effect on growth of F. graminearum and FHB severity reduction in a glasshouse experiment using chitosan and chitosan nanoparticles, but they did not evaluate DON contamination.
In our group, we demonstrated the efficacy of Bacillus velezensis RC218 and Streptomyces albidoflavus RC87B for reducing FHB severity and DON accumulation by F. graminearum under glasshouse and field experiments [24]. Taking into account the good results obtained during the present work with chitosan and F. graminearum in situ, we are developing further experiments combining the biological control agent with chitosan in order to improve the achieved results.
In conclusion, the present study showed the combined effects of chitosan and a W on growth and mycotoxin production by the three most important Fusarium species present on maize and wheat. Also, it was demonstrated that low-Mw chitosan with more than 70% DD at 0.5 mg/mL was able to significantly reduce growth rate and mycotoxin production on irradiated grains. It is important to remark that the effect was strain-specific, and, for this reason, these types of antifungal tests need to be made with at least two strains. Since mycotoxins are unavoidable contaminants in food and feed chains, their presence needs to be reduced in order to reduce their effects on human and animal health and to decrease the annual market losses caused by rejected cereals. In this scenario, pre/post-harvest (for F. graminearum) and post-harvest (for F. verticillioides and F. proliferatum) use of chitosan could be an important alternative. The use of chitosan is proposed as a post-harvest alternative treatment in maize in Argentina since permanent storage capacity is not increasing at the same rate as cereal production. Thus, a substantial portion of the harvest (17 million tons) is stored in temporary hermetic storage systems called silo-bags that remain in the field for long periods (over five months). Normally, these bags are filled with maize at 14% to 16% moisture content (wet basis) (0.72-0.8 aw) [51]. Pacin et al. [52] evaluated fumonisin contamination in maize stored in silo-bags and observed that, in maize stored for around 200 days following good agricultural practices, fumonisin contamination increased significantly. Regarding wheat grains, we propose the use of chitosan as a pre-and post-harvest strategy. As the global wheat production increased steadily in recent years, a substantial portion of the harvest is stored under natural conditions with no controlled facilities in many developed countries. Yuan et al. [53] observed that DON levels increased up to 30% after three months of storage. Thus, post-harvest control strategies are necessary, and chitosan could also be used. However, it is necessary to continue with further basic studies that could contribute to explaining the effect of applying this polymer on cereals.

Chitosan Solution
Low-viscosity chitosan was used (Fluka 50494; LVC; viscosity: ≤200 mPa·s). A stock solution was prepared by dissolving 10 g/L of chitosan in 1% acetic acid (AcH), and the solution was stirred for 24 hours at 28 • C. The pH of the chitosan solution was adjusted to 5.6 using NaOH in order to ensure that all the chitosan amino groups were positively charged [35,54]. Then, the solution obtained was autoclaved at 121 • C for 15 min, and maintained at 4 • C until use.

Chitosan Characterization
The viscosity-average molecular weight (Mw) of chitosan was determined using the intrinsic viscometric method using the Mark-Houwink-Sakurada equation [55]. The percentage of the chitosan amino groups (degree of deacetylation, DD) was determined using an infrared spectroscopy (Bruker Tensor 27) analysis, applying the following equation: where A 1655 is the absorbance at 1655 cm −1 of the amide I band and A 3450 is the absorbance at 3450 cm −1 of the hydroxyl band [37].

Fungal Strains
One F. verticillioides (M7075) strain and one F. proliferatum (RC2080) strain were used. Both strains were isolated from maize in Argentina [56,57] and were characterized using a polyphasic approach: morphological, biological, and genetic (amplified fragment length polymorphisms; AFLP). Also, fumonisin production capability was demonstrated. Moreover, two strains identified as F. graminearum sensu stricto, RC22-2 and RCFG6001, respectively, isolated from wheat spikes with Fusarium head blight (FBH) symptoms, were included in the present study. These strains were identified morphologically, by AFLP, and by partial sequencing of elongation factor gene (EF-1α); DON production capability was also demonstrated in previous studies [21,58]. The strains were deposited at the Department of Microbiology and Immunology, Universidad Nacional de Rio Cuarto culture collection (RC). Cultures were maintained in 15% glycerol at −80 • C (Table S1 in the Supplementary Materials).

Grains
Maize and wheat grains were gamma-irradiated (10-12 kGy) using a cobalt radiation source and were stored aseptically at 4 • C. Grains contained no fungal infection or contamination, but retained germinative capacity. The initial values of a W of the grains were 0.75 and 0.65 for maize and wheat, respectively. Irradiated maize and wheat grains were weighed and placed into sterile flasks and rehydrated to the required a W (0.99, 0.98, and 0.95 for maize, and 0.995, 0.99, and 0.98 for wheat) via addition of sterile distilled water using a moisture absorption curve. An appropriate aliquot of water was replaced by chitosan stock solution to give to the irradiated grains a final concentration (0.5, 1, and 2 mg/g). Flasks were subsequently refrigerated at 4 • C for 48 h with periodic shaking to allow absorption and equilibration. At the end of this period, the a W was checked with an Aqualab Series 3 (Decagon Devices, Inc, WA, USA). The rehydrated maize and wheat were placed in sterile 9-cm Petri dishes to form thin layers of grains (~20 g).
In order to discard fungal inhibition due to acetic acid in the chitosan solution, irradiated grains without chitosan (control AcH) were prepared for each a W level used. Those AcH control plates were prepared by adding the same volume of 1% AcH (pH adjusted to 5.6) used to prepare 2 mg/g of chitosan-amended wheat and maize grains.

Inoculation, Incubation, and Growth Assessment
All plates were inoculated with a 4-mm-diameter agar disc that was taken from the margin of a seven-day-old colony of each strain grown on synthetic nutrient agar at 25 • C [59]. The discs were transferred face-down onto the center of each plate. Inoculated Petri plates containing grains of the same a W were enclosed in plastic containers together with two beakers of NaCl-water solutions of the same a W as the treatments to maintain constant equilibrium relative humidity (ERH) inside the boxes. Containers were incubated at 25 • C for 28 and 21 days for irradiated maize grains and irradiated wheat grains, respectively.
Experiments were carried out in triplicate for each treatment. Assessment of growth was made daily during the incubation period, and two diameters of the growing colonies were measured at right angles to each other until the colony reached the edge of the plate. The radii of the colonies were plotted against time, and linear regression was applied in order to obtain the growth rate (mm/day) as the slope of the line. After the incubation period, three complete Petri plate cultures per treatment were sampled, dried at 50 • C for 24 h, and stored at −20 • C until toxin analysis (FB extraction from irradiated maize grains, and DON extraction from irradiated wheat grains).

Fumonisin Determination in Maize
Each sample was finely ground and mixed well. A sub-sample (15 g) was extracted with 40 mL of a mixture of acetonitrile-water (1:1 v/v) for 30 min using an orbital shaker (150 rpm) and then filtering the extracts through a paper filter (Nº 4, Whatman International Ltd, Maidstone, Kent, United Kingdom). An aliquot of extract (1000 µL) was taken and stored in an Eppendorf tube at −20 • C until HPLC analysis. were analyzed using a reversed-phase HPLC/fluorescence detection system. The HPLC system consisted of a Hewlett-Packard 1100 pump (Hewlett-Packard, Palo Alto, CA, USA) connected to a Hewlett-Packard 1046A programmable fluorescence detector and a data module Hewlett-Packard Kayak XA (HP ChemStation Rev. A.06.01). Chromatographic separations were performed on a stainless-steel, C 18 reversed-phase column (150 × 4.6 mm inner diameter (i.d.), 5 µm particle size; Luna-Phenomenex, Torrance, CA, USA) connected to Security Guard cartridge (4 × 3 mm i.d., 5 µm particle size; Phenomenex, Torrance, CA, USA) filled with the same phase. Methanol-0.1 M sodium dihydrogen phosphate (75:25, v/v) solution adjusted to pH 3.35 with orthophosphoric acid was used as the mobile phase, at a flow rate of 1.5 mL/min. Fluorescence of the fumonisin OPA derivatives was recorded at excitation and emission wavelengths of 335 and 440 nm, respectively. Fumonisins were measured as peak heights and compared with reference standard solutions (Sigma Chemical Co, St. Louis, MO, USA). A mixed acetonitrile-water (1:1, v/v) stock solution of FB 1 , FB 2 , and FB 3 containing 50 µg/mL of each toxin was prepared. Four mixed working calibrant solutions (0.25, 0.5, 1.0, and 2.0 µg/mL) were prepared by diluting an aliquot of the stock solution with the appropriate volume of acetonitrile-water (1:1, v/v). The retention times of FB 1 , FB 3 , and FB 2 were 7.5, 16.7, and 18.5 min, respectively. Appropriate dilutions of standards and/or sample extracts were made with acetonitrile-water (1:1). The detection limit (LOD) of the analytical method for the three fumonisins was 1 µg/g based on the signal-to-noise ratio 3:1.

Deoxynivalenol Determination in Wheat
The DON analysis was done using a modified version of that originally reported by Cooney et al. [61]. Each sample was finely ground and mixed well. A sub-sample (15 g) was extracted by mixing with acetonitrile-methanol (14:1; 40 mL), shaken for 2 h, and then filtered through filter paper (Whatman N • 1, Whatman International Ltd, Maidstone, Kent, United Kingdom). A syringe was plugged with glass wool and dry-packed with alumina/carbon (20:1; 300 mg) to form a mini-cleanup column. A 2-mL aliquot of extract was applied to the column and allowed to drain under gravity, and the eluent was collected. The column was washed with 500 µL of a mixture of acetonitrile-methanol-water (80:5:15 v/v), and the combined eluant was evaporated to dryness using N 2 at 50 • C. The cleaned-up residue was dissolved in 500 µL of methanol-water (5:95 v/v) and stored until HPLC analysis. The HPLC system consisted of a Hewlett-Packard model 1100 pump (Palo Alto, CA) connected to a Hewlett-Packard 1100 Series variable wavelength detector and a data module Hewlett-Packard Kayak XA (HP ChemStation Rev. A.06.01). Chromatographic separations were performed on a Luna™ C 18 reversed-phase column (100 × 4.6 mm, 5 µm particle size) connected to a guard column, SecurityGuard™ (4 × 3.0 mm), filled with the same phase. The mobile phase consisted of methanol-water (12:88, v/v), at a flow rate of 1.5 mL·min −1 . The detector was set at 220 nm with an attenuation of 0.01 AUFS (absorbance units full scale). The injection volume was 50 µL. Quantification was relative to external standards of DON (Sigma-Aldrich Co. St Louis, MO) of 1 to 4 µg/mL in methanol-water (5:95). The quantification (LOD) limit was 5 ng/g.

Statistical Treatment of Results
The growth rates, lag phases, and mycotoxin concentrations were evaluated by analysis of variance (ANOVA) to determine the effect of chitosan doses, a W , Fusarium species, and two-and three-way interactions. When the analysis was statistically significant, the post hoc Tukey´s multiple comparison procedure was used for separation of the means. Statistical significance was judged at the level p ≤ 0.001. All the analyses were done using SigmaStat for Windows Version 2.03 (SPSS Inc.) Supplementary Materials: The following are available online at http://www.mdpi.com/2076-0817/8/1/29/s1, Table S1: Characteristics of the Fusarium strains used.
Author Contributions: M.L.R. and S.N.C. designed the entire experiment. V.G.L.Z. was responsible for carrying out the entire experimental work. M.J.N. and V.G.L.Z. performed mycotoxin analysis. E.C. and V.G.L.Z. were responsible for data analysis. E.C. and M.L.R. wrote the paper, and M.J.N., V.G.L.Z., and S.N.C. revised the manuscript.