Introduction

Chitosan is the collective name given to a group of partially and fully deacetylated derivatives of chitin, a long-chain polymer of N-acetylglucosamine [1]. Chitosan has been widely used as an antimicrobial agent in the food, biomedicine, agriculture, wastewater treatment, and cosmetics industries due to its low cost, stability for long-term usage, high biodegradability, nontoxicity to mammalian cells, antitumor activity, and potent biocidal activity against a broad spectrum of pathogenic microorganisms [2–6]. However, the action of chitosan is highly affected not only by intrinsic factors, including the degree of deacetylation, the density of positive charge, and the molecular weight of chitosan polymers, but also by environmental conditions including chemical compositions, ionic strength, and pH in solution [4, 6, 7].

Although the mode of action for chitosan is still largely unknown, some models have been proposed based on physiological observations. The maximum antimicrobial activity of chitosan is often observed to occur slightly below its pKa value of 6.2, which induces polycationic chitosan by protonation of the amino groups [4, 7, 8]. This suggests that electrostatic interactions between the positively charged chitosan molecule and the negatively charged microbial cell surface might lead to internal osmotic imbalances, leakage of intracellular constituents, and ultimately death of the cell [3, 4]. Another possible model for the action of chitosan is the interference with cell stability and integrity by chelation of crucial metal ions associated with the cell wall [3, 4], leading to membrane permeability and cell death.

The metal chelator ethylenediaminetetraacetatic acid (EDTA) has been widely used as an antimicrobial agent in cleaners, agriculture, and food processing [9]. It exerts a synergistic or potentiating action with other antimicrobial compounds, such as antibiotics, chitosan, and quaternary ammonium compounds [10, 11]. However, the mechanism underlying the synergistic or additive properties of EDTA with antimicrobial compounds is largely unknown. As in the model for chitosan, the action of EDTA may involve chelation and the deprivation of essential divalent cations required for the growth of microorganisms [10, 11].

Chitosan and its derivatives have been extensively explored as methods for controlling plant diseases because they possess not only direct antimicrobial activity against phytofungi but also the ability to elicit defense responses in host plants [12]. Chitosan treatment by soil amendment and foliar application has shown some success in controlling or preventing plant disease or triggering plant defenses against phytopathogens in tomato, wheat, soybean, maize, peas, lentils, and rice plants [12, 13]. Moreover, the simple application of chitosan as a seed coating agent has been reported to accelerate seed germination and improve resistance to infection by phytopathogens in some crops [14]. However, the potential uses of chitosan as an antimicrobial agent still need to be further investigated for specific species of pathogens and certain environmental conditions.

Bakanae, commonly known as foolish seedling, is a destructive disease of rice plants caused by infection by Fusarium fujikuroi (Gibberella fujikuroi mating population C), which is widely distributed in all the rice growing areas including Asia, Africa, and North America. Incidences of bakanae disease and substantial losses of rice yield have been increasing in South Korea [15, 16]. As a disease management strategy, chemical fungicides, such as prochloraz and benomyl, have been used to control Bakanae disease. However, this is not effective since numerous fungicide-resistant strains of F. fujikuroi have arisen in the last decades [16].

In this study, we compared the antimicrobial activity of COS with that of EDTA to get a better understanding of the mode of action, and to evaluate these as eco-friendly fungicides for control of F. fujikuroi causing rice Bakanae disease. COS exhibited rapid fungicidal activity with pore-formation and leakage of intracellular constituents of hyphal cells, whereas EDTA showed fungistasis activity without significant changes in cellular viability and morphology. The actions of COS and EDTA were mediated differentially by Ca2+ and Mn2+. Moreover, COS and EDTA exhibited additive antimicrobial effects. This suggests that the application of COS together with EDTA might be effective in managing Bakanae disease caused by F. fujikuroi.

Materials and Methods

Materials

Chitosan oligosaccharides (COS) was prepared as described [5]. Briefly, high molecular weight chitosan of approximately 1900 kDa and 98.5 % degree of deacetylation (St. Louis, MO, USA) was dissolved in 2 % acetic acid to make 2 % chitosan solution and hydrolyzed by recombinant chitosanase (1 U/ml) (Kyowa Chemical Ltd., Kagawa, Japan). The enzyme hydrolyzed product was further fractionated by gel-filtration chromatography using Bio Gel-P4 (Bio-Rad, Richmond, CA, USA). Then, a major carbohydrate-positive fraction consisting of a mixture of fully deacetylated (COS)3–5 was obtained and used in this study. Disodium ethylenediaminetetraacetatic acid (Na2EDTA), CaCl2, MgCl2, MnCl2, and ZnCl2 were purchased from Sigma chemical company (St. Louis, MO, USA).

Fungal Strains

Fusarium fujikuroi strains isolated in South Korea were obtained from the Korean Agricultural Culture Collection (KACC), as listed in Table 1. The liquid media used for this study were potato dextrose broth (PDB) medium (Difco Laboratories, Detroit, MI, USA), and the solid medium was prepared by adding 1.2 % (w/v) agar to PDB medium. For isolation of fungal spores, an agar plug containing freshly growing mycelia was placed in the center of carrot agar medium [17] and incubated for 6 days at 24 °C with a 12-h photoperiod under fluorescent light.

Table 1 Antimicrobial activities of COS and EDTA to 20 strains of F. fujikuroi Nirenberg isolated in South Koreaa
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Antimicrobial Assay

The inhibitory activities of COS and EDTA on fungal spore germination were determined by microspectrophotometry of liquid cultures as described [18]. Briefly, various concentrations of COS or EDTA were mixed with 104 fungal spores in 100 μl of 1 × PDB medium. To analyze the effect of divalent cations on COS and EDTA activity, various concentrations of COS or EDTA were mixed at the indicated concentrations of CaCl2, MnCl2, MgCl2, and ZnCl2 in 100 μl of 0.5x PDB medium containing 104 fungal spores. We should note that inhibitory activities of COS and EDTA are negative correlations with the strength of PDB medium in a dose-dependent manner. The OD600nm values of the microplate wells were measured after incubation at 24 °C for 2 days and are represented as percent growth. Minimum inhibitory concentrations required to inhibit the growth of 50 and 90 % (MIC50 and MIC90) were determined from dose–response curves.

Cell Death Analysis

An inoculum of 106 F. fujikuroi spores in suspension was added to 10 ml of PDB medium and incubated for 1 day at 24 °C on an orbital shaker to allow germination. Aliquots of germinating fungal cells were treated with inhibitory concentration of COS (40 μg/ml), EDTA (3.2 mM), or their combination (40 μg/ml and 3.2 mM), for 0, 5, 15, 30, and 60 min at 24 °C. Samples were then diluted with sterile water and plated on PDA plates. CFU was determined after incubation on PDB agar plate for 2–3 days at 24 °C. Three replicates were used for each treatment. Scanning electron microscopy analysis was carried out as described [18].

Statistical Analysis

Data are represented as the mean ± standard deviation (SD). Mean values among experiments were compared by Tukey’s multiple comparison test at P < 0.05. Analysis of variance (ANOVA) was performed using OriginPro software version 8.5 (OriginLab Co., USA).

Results and Discussion

Antimicrobial Activities of COS and EDTA

Twenty strains of F. fujikuroi isolated in South Korea were obtained from KACC (Table 1). Some of them were classified as strains resistant to chemical fungicides used in the management of Bakanae disease, such as prochloraz and benomyl. In a microplate-based antimicrobial assay, both COS and EDTA had potent inhibitory effects on spore germination of F. fujikuroi strains, irrespective of fungicide resistance and sensitivity (Table 1). The MIC90 values of COS for all tested strains were 40 μg/ml, and MIC50 values ranged from 19.5 ± 2.5 to 21.5 ± 2.5 μg/ml. These activities are similar to those of low molecular weight chitosan on 8 other phytofungi and Candia albicans [19], but much higher than those of chitin and crude chitosan against 46 different fungi [2]. Similarly, EDTA exhibited similar inhibitory activity against all tested strains with MIC50 values ranged from 0.55 ± 0.00 to 0.62 ± 0.09 mM and MIC90 values of 1.5 mM, respectively. These results suggest that both COS and EDTA might be good candidates for the management of F. fujikuroi strains, including those showing fungicide resistance.

Fungicidal or Fungistatic Activity of COS and EDTA

To investigate whether COS and EDTA influence the viability of F. fujikuroi cells, a time-killing curve was performed after treatment with inhibitory concentrations of COS, EDTA, or both in combination. Representative data from experiments using 20 strains are shown in Fig. 1. The hyphal growing cells treated with EDTA showed no significant (P > 0.05) change in their viability for up to 1 h compared to their viability in the water control. Under the same conditions, treatment with COS alone or COS combined with EDTA induced rapid cell death within 5 min, and viability was completely lost after 30 min of incubation. However, the minimum fungicidal concentrations between COS alone and combinations of COS and EDTA were not significantly (P > 0.05) different. This indicates that EDTA acts as fungistatic agent by reversible cell growth arrest, whereas COS acts as fungicidal agent to F. fujikuroi.

Fig. 1
figure 1

Effects of COS and EDTA on cell viability. Hyphal growing cells of F. fujikuroi were incubated with water as a control, 40 μg/ml COS, 3.2 mM EDTA, or COS and EDTA together for 5, 15, 30, and 60 min. The cells were diluted with sterile water and plated on PDB agar plates. CFU were determined after incubation for 2–3 days at 24 °C. Error bars represent the standard error of the mean (n = 3)

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To further investigate the fungicidal or fungistatic effect, morphological alterations of F. fujikuroi cells were examined by scanning electron microscope after treatment with inhibitory concentration of COS and EDTA. Hyphal growing cells treated with EDTA showed a slight wrinkling of the surface in some cells, but no visible difference was observed in the majority of cells compared to the water control (Fig. 2). However, when the cells were treated with COS, large or small aggregates on the cell wall caused by leakage of cytoplasmic contents were observed in most cells. Furthermore, a higher magnification view revealed that approximately 70 % of cells contained large pores on the cell surface and lethal damage to hyphae. Again, these results suggest that the primary mode of action of COS is likely pore-formation on cell membranes which subsequently leads to cellular leakage and cell death, whereas EDTA acts as a fungistatic agent through reversible growth inhibition in F. fujikuroi.

Fig. 2
figure 2

Effects of COS and EDTA on the morphology of F. fujikuroi cells. Hyphal growing cells of 3 F. fujikuroi strains (KACC No. 44004, 44018, and 44021) were treated with water (a, d), 3.2 mM EDTA (b, e) and 40 μg/ml COS (c, f) for 30 min at 24 °C in PBD medium. The cells were fixed and processed for scanning EM analysis. Similar results were observed from the experiment with three strains, and representative data were shown. Scale bars 10 μm (a–c) and 0.2 μm (d–f)

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Effect of Divalent Cations on COS and EDTA Activity

It has been reported that COS and EDTA activity is affected by ionic strength and metal ions in the medium [3, 4]. Thus, we examined the effect of physiologically abundant divalent cations, such as Ca2+, Mg2+, Mn2+, and Zn2+, on COS and EDTA activity. Since all divalent cations used in this experiment were chloride salts, the effect of specific divalent cations could be addressed. In preliminary experiments, we determined the optimum concentrations of cations which resulted in no significant effect on growth. These concentrations were then used in this experiment.

The addition of Mn2+, Mg2+, and Zn2+ to the assay mixture did not affect COS activity against F. fujikuroi (Table 2). No significant (P > 0.05) changes in the IC50 and IC90 values were shown compared to those of non-treated controls. However, dose-dependent reductions in COS activity were observed upon addition of Ca2+. The IC50 values were increased threefold (P < 0.05) and more than fivefold (P < 0.05) by adding 2.5 mM and 5 mM CaCl2, respectively. The IC90 values were also increased more than fourfold by the addition of Ca2+. Upon addition of Mg2+ and Zn2+ to the assay mixture, no significant (P > 0.05) changes in the EDTA activity were observed compared to that of the non-treated control (Table 2). However, EDTA activity was mostly suppressed by addition of Mn2+ to the assay mixture. Both the IC50 and IC90 values of EDTA were increased more than 18-fold by the addition of 2.5 mM Mn2+. Unlike with COS, the effect of Ca2+ on EDTA activity was much less than that of Mn2+ with a twofold increase in the IC90 value.

Table 2 Effect of divalent cations on COS and EDTA activitya
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It is well known that a number of metal cations are important for many cellular responses, including ion homeostasis, signaling pathways, morphogenesis, and cell wall biogenesis upon environmental stresses in fungal cells. For example, activation of the calcineurin and PKC1 pathway by addition of Ca2+ suppresses the growth arrest or defects of cell lysis upon cell wall stress in yeast [21]. Indeed, loss-of-function mutations in this pathway, such as Δbck1 and Δcrz1, resulted in increased sensitivity to chitosan treatment [22, 23]. Furthermore, deletion of the zinc-responsive transcription factor Zap1p or addition of the zinc chelator 1,10-phenanthroline results in increased toxicity of pleiotropic drug diclofenac in yeast [20, 24]. Thus, COS and EDTA action might be mediated through interactions with specific molecules related to Ca2+ and Mn2+ homeostasis in F. fujikuroi cells. However, their specificity to divalent cations and action mechanism are likely different depending on microorganism or chemical properties of chitosan. Unlike Ca2+-mediated COS action in this study, the antibacterial activity of chitosan microspheres to Escherichia coli was most effectively inhibited by the addition of Mg2+ into a medium [4]. Otherwise, Chung et al. [25] reported that addition of Zn2+ into a medium inhibited the antibacterial activity of shrimp chitosan against E. coli and Staphylococcus aureus most effectively compared to Ca2+, Mg2+, and Ba2+. Similarly, the antifungal activity of EDTA against yeast and some filamentous fungi was highly inhibited by the addition of Zn2+ to the medium compared to negligible effect of Mn2+ and other ions including Fe3+, Cu2+, and Mg2+ [20].

Since the emergence of fungicide-resistant F. fujikuroi strains causing Bakanae rice disease has gradually increased in South Korea [16], eco-friendly fungicides are urgently needed to prevent or delay the development of resistant strains. COS and EDTA are known to be biosafe and biocompatible antimicrobial agents. In this study, we showed that both have potent and similar growth inhibitory effects on F. fujikuroi strains, irrespective of fungicidal resistance or sensitivity. This suggests that the target molecules for COS and EDTA on fungal cell are different from those for chemical fungicides. Moreover, their modes of actions against F. fujikuroi are also different. Although we evaluated the effect of two chelating agents, COS and EDTA, against F. fujikuroi in this study, further studies on the different antimicrobial action of chelating agents may provide more effective and customized strategies to control plant diseases caused by phytofungi.