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Appl Environ Microbiol. Sep 2009; 75(17): 5563–5569.
Published online Jul 17, 2009. doi:  10.1128/AEM.00711-09
PMCID: PMC2737905

Sporicidal Activity of Synthetic Antifungal Undecapeptides and Control of Penicillium Rot of Apples [down-pointing small open triangle]

Abstract

The antifungal activity of cecropin A(2-8)-melittin(6-9) hybrid undecapeptides, previously reported as active against plant pathogenic bacteria, was studied. A set of 15 sequences was screened in vitro against Fusarium oxysporum, Penicillium expansum, Aspergillus niger, and Rhizopus stolonifer. Most compounds were highly active against F. oxysporum (MIC < 2.5 μM) but were less active against the other fungi. The best peptides were studied for their sporicidal activity and for Sytox green uptake in F. oxysporum microconidia. A significant inverse linear relationship was observed between survival and fluorescence, indicating membrane disruption. Next, we evaluated the in vitro activity against P. expansum of a 125-member peptide library with the general structure R-X1KLFKKILKX10L-NH2, where X1 and X10 corresponded to amino acids with various degrees of hydrophobicity and hydrophilicity and R included different N-terminal derivatizations. Fifteen sequences with MICs below 12.5 μM were identified. The most active compounds were BP21 {Ac,F,V} and BP34 {Ac,L,V} (MIC < 6.25 μM), where the braces denote R, X1, and X10 positions and where Ac is an acetyl group. The peptides had sporicidal activity against P. expansum conidia. Seven of these peptides were tested in vivo by evaluating their preventative effect of inhibition of P. expansum infection in apple fruits. The peptide Ts-FKLFKKILKVL-NH2 (BP22), where Ts is a tosyl group, was the most active with an average efficacy of 56% disease reduction, which was slightly lower than that of a commercial formulation of the fungicide imazalil.

The discovery of antimicrobial compounds to treat plant diseases of economical importance in agriculture remains a major scientific challenge (1). Antimicrobial peptides are being considered as a good alternative to current fungicides and a great deal of scientific effort has been invested in studying their application in plant disease control (29, 34, 35).

Antimicrobial peptides have been reported to display interesting activities against pathogenic microbes that are resistant to conventional antibiotics and to exhibit a broad spectrum of activity against bacteria, fungi, enveloped viruses, parasites, and tumor cells (7-10, 19, 20, 40, 49). The mechanism of action of these peptides against fungi consists of cell lysis by binding to the membrane surface and disrupting its structure, interference with the synthesis of essential cell wall components, or interaction with specific internal targets (12, 13, 15, 23, 29).

Despite their good lytic activity, major concerns about the use of antimicrobial peptides as pesticides in plant protection are the high production cost associated with synthetic procedures and their low stability toward protease degradation. Several design strategies have been devised in order to find shorter and more stable peptides, while maintaining or increasing the activity with a low cytotoxicity. These strategies include the juxtaposition of fragments of natural antimicrobial peptides, the modification of natural peptides, and the de novo design of sequences maintaining the crucial features of native antimicrobial peptides (2, 3, 11, 24, 32, 38, 42). However, the process involved in the development of lead candidates is time consuming and limited by the number of individual compounds that can be synthesized. Combinatorial chemistry has allowed the rapid preparation of synthetic libraries and their screening has led to the identification of peptides with high activity against selected phytopathogenic bacteria and fungi (4, 26, 27, 33).

During our current research oriented to the development of new antimicrobial agents for use in plant protection, we designed linear undecapeptides (CECMEL11) derived from the cecropin A-melittin hybrid peptide WKLFKKILKVL-NH2 (Pep3) (5, 17). Using a combinatorial approach, we identified peptides with high activity against plant pathogenic bacteria, such as Erwinia amylovora, Xanthomonas vesicatoria, and Pseudomonas syringae, and with low susceptibility to protease degradation (4, 5).

In order to broaden the study, we decided to test the CECMEL11 peptides against the plant pathogenic fungi Fusarium oxysporum, Aspergillus niger, Rhizopus stolonifer, and Penicillium expansum. The fungus F. oxysporum causes Fusarium wilt in more than a hundred species of plants, and it is an important pathogen in horticultural crops (44). Several Rhizopus and Penicillium species cause soft rot and blue mold rot, respectively, which are important postharvest diseases in stone and pome fruits (6, 18, 22, 39). Apart from the economic losses, Aspergillus and Penicillium species are also of interest from a public health point of view due to the production of mycotoxins (45, 47). The importance of Penicillium species in the postharvest of fruits emphasizes the interest to develop antimicrobial peptides to control this fungus.

Taking into account the relevance of these pathogens, the aim of the present study was the analysis of the antifungal activity profile of the CECMEL11 peptides in order to identify sporicidal sequences against the above fungi. As a proof of concept, the feasibility of using such peptides to protect fruits from fungal spoilage was evaluated using a P. expansum/apple model.

MATERIALS AND METHODS

Peptide synthesis.

All peptides were synthesized as previously described by the solid-phase method using 9-fluorenylmethoxycarbonyl (Fmoc)-type chemistry, tert-butyloxycarbonyl side chain protection for Lys and Trp, and tert-butyl for Tyr (4, 5, 17). Fmoc-Rink-4-methylbenzhydrylamine resin (0.64 mmol/g) was used as solid support to obtain C-terminal peptide amides and 4-hydroxymethylphenoxypropionic acid polyethylene glycol-polystyrene resin (0.23 mmol/g) to synthesize the C-terminal peptide acid. Couplings of the Fmoc amino acids (3 eq) were mediated by N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HBTU) (3 eq) and N,N-diisopropylethylamine (DIEA) (3 eq) in N,N-dimethylformamide (DMF) and monitored by a ninhydrin test. The Fmoc group was removed by treating the resin with a mixture of piperidine-DMF (3:7). Acetylation was performed by treatment with acetic anhydride-pyridine-CH2Cl2 (1:1:1). Tosylation was carried out by treatment with p-toluenesulphonyl chloride (40 eq) and DIEA (80 eq) in CH2Cl2-N-methyl-2-pyrrolidinone (CH2Cl2-NMP) (9:1). Benzoylation was performed by treatment with benzoyl chloride (40 eq) and DIEA (80 eq) in CH2Cl2-NMP (9:1). Benzylation was achieved by treatment with benzyl bromide (40 eq) and DIEA (80 eq) in CH2Cl2-NMP (9:1). Peptides were cleaved from the resin with trifluoroacetic acid-H2O-triisopropylsilane (95:2.5:2.5) and were obtained with >90% high-performance liquid chromatography purity. Electrospray ionization mass spectrometry was used to confirm peptide identity.

Fungal strains and growth media.

The following plant pathogenic fungal strains were used: Penicillium expansum EPS 26 (INTEA, University of Girona), Fusarium oxysporum f. sp. lycopersici FOL 3 race 2 (ATCC 201829; American Type Culture Collection), Aspergillus niger (CECT 2694; Colección Española de Cultivos Tipo), and Rhizopus stolonifer (CECT 2344). The strains were cultured on potato dextrose agar (PDA) plates (Difco) using aseptic procedures to avoid contamination. Conidia from fungal mycelium (except for F. oxysporum) were obtained from 5-day-old PDA cultures of the fungus incubated at 25°C. The inoculum was prepared by scraping spore material from the culture surfaces with a wet cotton swab and resuspending it in distilled water containing 0.5% of Tween 80. Microconidia of F. oxysporum were obtained from 1-week-old potato dextrose broth (PDB) cultures (Oxoid) of the fungus incubated at 25°C in the dark in a rotary shaker at 125 rpm. After incubation, the culture was filtered through several layers of sterile cheesecloth to eliminate macroconidia and mycelial growth of the fungus. Then, the effluent was centrifuged at 8,000 × g for 20 min at 4°C, and the pellet was resuspended in sterile water. The concentration of conidia was determined using a hemacytometer and adjusted to 104 conidia ml−1 for F. oxysporum, 103 conidia ml−1 for P. expansum and A. niger, and 102 conidia ml−1 for R. stolonifer.

Antifungal activity.

The lyophilized peptides listed in Table Table11 were solubilized in sterile Milli-Q water to a final concentration of 1 mM and filter sterilized through a 0.22-μm-pore filter (Sartorius). For MIC assessment, stock solutions of peptides were prepared at different concentrations. Antifungal activity was tested on F. oxysporum, P. expansum, A. niger, and R. stolonifer. Twenty microliters of each stock solution was mixed in a microtiter plate well with 80 μl of the corresponding suspension of the fungal pathogen and 100 μl of double-concentrated PDB to a total volume of 200 μl containing 0.003% (wt/vol) of chloramphenicol. The final peptide concentrations assayed were 100, 50, 25, 20, 15, 12.5, 10, 7.5, 5, 2.5, 1.2, 0.6, and 0.3 μM. Three replicates for each strain, peptide, and concentration combination were used. Positive controls contained water instead of peptide, and negative controls contained peptides without the fungal pathogen. Microbial growth was automatically determined by optical density measurement at 600 nm (Bioscreen C; Labsystems, Helsinki, Finland). Microplates were incubated at 20°C with 1 min/9 min on/off shaking cycles, and the measurements were done every 2 h for 6 days. The MIC was taken as the lowest peptide concentration without growth at the end of the experiment.

TABLE 1.
Antifungal activity and hemolytic activity of selected linear undecapeptides

The 125-member peptide library was tested for antifungal activity on P. expansum at final concentrations of 25, 12.5, and 6.25 μM, as described above.

Fungicidal activity against nongerminated F. oxysporum and P. expansum conidia.

Stock solutions of peptides were prepared at different concentrations. Microconidial suspensions of F. oxysporum and P. expansum were adjusted to 8 × 106 conidia ml−1, as described above. The test tubes contained 190 μl of the conidial suspension, 100 μl of the corresponding peptide concentration, and 710 μl of 50-fold diluted PDB. The final peptide concentrations assayed were 20, 15, 10, 5, and 2.5 μM for F. oxysporum and 25, 12.5, and 6.25 μM for P. expansum. After mixing, the test tubes were incubated for 35 min (F. oxysporum) or 180 min (P. expansum) at 25°C and 10-fold serially diluted, and aliquots of 100 μl from suitable dilutions were spread in triplicate on PDA plates. The sample dilution and additional dilution into the total amount of medium contained on the PDA plate were sufficient to prevent residual activity of the peptide. Viable colonies were counted after incubation for 3 days at 25°C. Values were expressed as percentages of survival from the start of the experiment.

Effect of peptides on Sytox green uptake by F. oxysporum conidia.

A suspension of F. oxysporum conidia in 20-fold-diluted PDB (180 μl, 5 × 105 conidia ml−1) was dispensed into microtiter plate wells. Subsequently, 20 μl of the corresponding peptide stock solution, as described above for fungicidal activity, was added, and the plates were incubated for 24 h at 25°C. Then, Sytox green was added to a final concentration of 0.2 μM, and the plates were incubated for 1 h at 25°C to stabilize the fluorescence. Sytox green uptake was determined by fluorometric measurement with a microplate reader (Varioskan, Ascent FL; Labsystems, Finland), recording the fluorescence spectra between 510 and 580 nm. The controls were prepared with water instead of peptide. Three replicates for each treatment were prepared. The fluorescence increase with respect to that of the control was calculated at 528 nm. Dose-response curves for each peptide were generated by plotting the fluorescence increase against the peptide concentration.

Hemolytic activity.

The hemolytic activity of peptides was evaluated at 150 μM. Hemoglobin release from erythrocyte suspensions of fresh human blood (5%, vol/vol) was determined using the absorbance at 540 nm, as previously described (17).

Inhibition of P. expansum infections in apple fruits.

Golden apples were collected at harvest time from a commercial orchard near Girona, Spain, washed, surface-disinfected by immersion for 1 min in a diluted solution of sodium hypochlorite (1% active chlorine), and washed two times by immersion in distilled water. The excess of water was subsequently removed under airflow. The fruits were wounded with a cork borer, making six wounds per fruit of approximately 5 mm2 and a 10-mm depth.

Preliminary tests were performed to evaluate the phytotoxicity and effective concentrations, using treated-uninoculated, nontreated-inoculated, and treated-inoculated treatments. The tests were carried out at 200 and 500 μM peptide concentrations.

For the main trial, peptide treatments were applied by depositing 50 μl of a 300 μM solution in each wound. Appropriate controls consisted of nontreated fruits and of fruits treated with the reference fungicide imazalil at the recommended concentration (375 μM Fungaflor [20%, wt/vol]; Janssen Pharmaceutica NV, Berse, Belgium). After the peptide or fungicide was completely absorbed, the wounds were inoculated with 20 μl of a P. expansum EPS26 spore suspension at 103 conidia ml−1. Three replicates of three fruits per replicate were prepared for each treatment. The apples were placed in polystyrene tray packs that were fit into boxes and maintained at 20°C. The lesion diameter was measured after 11 days of pathogen inoculation. The experiment was repeated twice. The data were analyzed using the general linear model procedure of the Statistical Analysis System (SAS version 9.1.2, 2009). To correct variance heterogeneity, the values were arcsine transformed before the analysis. A Tukey multiple range test (P = 0.05) was conducted to separate the means of the lesion diameter values for individual treatments in both experiments.

RESULTS

Evaluation of the antifungal activity of a set of CECMEL11 undecapeptides.

In a first screening, a set of 15 peptides was studied (Table (Table1).1). They are C-terminal amide 11-residue sequences except for BP1, which is a C-terminal acid peptide. Peptides BP8 to BP11 are derivatized at the N termini with an acetyl (Ac), tosyl (Ts), benzoyl (Bz), or benzyl (Bn) group, respectively. Analogues BP13 to BP20, BP33, and BP76 differ on the residues at positions 1 and 10, including amino acids with various degrees of hydrophobicity and hydrophilicity.

These peptides were screened in vitro for antifungal activity against F. oxysporum, P. expansum, A. niger, and R. stolonifer (Table (Table1).1). All sequences exhibited antifungal activity (MIC < 25 μM) against at least two pathogens. F. oxysporum was particularly sensitive to the peptides. Thirteen out of 15 sequences were more active against F. oxysporum than Pep3 with MICs below 2.5 μM. BP33 was the most active peptide (MIC of 0.3 to 0.6 μM), four sequences (BP15, BP16, BP20, and BP76) displayed antifungal activity within 0.6 and 1.2 μM, and eight peptides showed MICs ranging from 1.2 to 2.5 μM. The benzoylated peptide BP10 was slightly less active (MIC of 5.0 to 7.5 μM), while the C-terminal acid peptide BP1 displayed the lowest activity (MIC of 20 to 25 μM).

In the case of P. expansum, three peptides exhibited antifungal activity with MICs below 20 μM. Among them, analogues BP8 and BP15 were the most active (MIC of 7.5 to 10 and 12.5 to 15 μM, respectively), displaying higher activity than Pep3 (MIC of 15 to 20 μM). Peptides BP1 and BP16 were inactive up to a concentration of 100 μM. Among the peptides active against A. niger, four sequences showed antifungal activity below 20 μM. The best activity was observed for peptide BP15, being as active as Pep3 (MIC of 5.0 to 7.5 μM). R. stolonifer was the least sensitive fungi. Only BP15 exhibited an antifungal activity below 20 μM, being more active (MIC of 7.5 to 10 μM) than Pep3 (MIC of 10 to 12.5 μM). Four sequences were not active against this fungus (MIC > 100 μM), including the C-terminal acid peptide BP1 and the N-terminal-derivatized peptides BP8, BP10, and BP11.

The toxicity of this peptide set to red blood cells is included in Table Table1.1. When tested at 150 μM, two of these peptides (BP1 and BP16) were not hemolytic, six sequences showed a low to moderate level of hemolysis (<50%), and seven were more hemolytic (>50%). Interestingly, BP15, which displayed a high activity against all fungi tested, exhibited a low hemolysis (16%).

The fungicidal activities of the most active peptides, BP15, BP20, BP33, and BP76, were evaluated against F. oxysporum by comparing the survival of the conidia after 35 min of exposure at different peptide concentrations (Fig. (Fig.1).1). Pep3 was included for comparison purposes. At concentrations around the MIC (<5.0 μM), all peptides exhibited a similar behavior, whereas an increase of peptide concentration led to a different logarithmic pattern for each sequence. BP76, BP33, and BP20 were more potent than Pep3. Notably, BP33 was the most sporicidal, causing a 3- to 4-log survival reduction at 10 to 20 μM in 35 min.

FIG. 1.
Sporicidal activity of peptides Pep3 ([filled triangle]), BP15 (○), BP20 ([filled square]), BP33 ([open triangle]), and BP76 ([open diamond]) on F. oxysporum conidia (solid lines) and of peptides BP21 (□) and BP22 ([filled lozenge]) on P. expansum conidia (dashed lines). ...

The membrane permeation of F. oxysporum conidia by peptides Pep3, BP15, BP20, BP33, and BP76 was studied by determining the fluorescence increase observed on suspensions of the conidia after treatment with different peptide concentrations and Sytox green (Fig. (Fig.2).2). In all cases, fluorescence increased rapidly with peptide concentration, following a dose-response saturation relationship. The maximum values of fluorescence were achieved at 10 to 20 μM. BP20 was the most potent peptide in terms of the fluorescence-increase response, displaying a higher value than Pep3. Moreover, conidia survival was plotted against fluorescence increase (Fig. (Fig.3).3). Interestingly, survival percentage correlated significantly with Sytox green uptake (y = 100.3 − 35.46x; R2 = 0.88), indicating that probably membrane disruption is the primary mechanism of action.

FIG. 2.
Effect of peptides Pep3 ([filled triangle]), BP20 ([filled square]), BP33 ([open triangle]), BP15 (○) or BP76 ([open diamond]) on Sytox green uptake by F. oxysporum conidia. Conidia were incubated with peptides in 20-fold-diluted PDB at 25°C for 24 h; then, Sytox ...
FIG. 3.
Relationship between sporicidal activity and Sytox green uptake in F. oxysporum conidia treated with peptides Pep3 ([filled triangle]), BP20 ([filled square]), BP33 ([open triangle]), BP15 (○), or BP76 ([open diamond]).

Evaluation of a 125-member peptide library against P. expansum.

The general structure of the peptide library was R-X1KLFKKILKX10L-NH2, where X1 and X10 corresponded to amino acids with various degrees of hydrophobicity and hydrophilicity and R included different N-terminal derivatizations. The library was designed by combining five variations at each R, X1, and X10 position as follows: at the R position, H, Ac, Ts, Bz, or Bn; at the X1 position, Lys, Leu, Trp, Tyr, or Phe; and at the X10 position, Lys, Val, Trp, Tyr, or Phe (4). Library members are denoted using braces, such as {R,X1,X10}, which define the variations at each R, X1, and X10 position. This library was screened in vitro for antifungal activity against P. expansum (Fig. (Fig.4).4). Fifty-four sequences displayed activity below 25 μM. In general, peptides with Ac at the R position and Val at the X10 position were associated with high activity. Peptides BP21 {Ac,F,V} and BP34 {Ac,L,V} were the most active with MICs below 6.25 μM. Thirteen sequences showed moderate activity (MICs of 6.25 to 12.5 μM), and 39 peptides displayed low activity (MICs of 12.5 to 25 μM). The fungicidal activities of BP21 and BP22 {Ts,F,V} against P. expansum were evaluated (Fig. (Fig.1).1). These peptides were sporicidal, but a longer exposure (180 min) was necessary to kill the conidia, compared to that for F. oxysporum (35 min). The hemolysis of the peptides with a MIC of <25 μM ranged from 35 to 96% at 150 μM. Peptides BP21, BP22, and BP34, which exhibited high antifungal activity, showed 85, 73, and 45% hemolysis at this concentration, respectively. Although these hemolytic activities are significant, they are similar to that observed for the reference fungicide imazalil, which ranges from 53 to 77% at 150 μM.

FIG. 4.
MICs of the 125-member peptide library against P. expansum in full-strength PDB. Amino acids with various degrees of hydrophilicity and hydrophobicity (X1 and X10) and N-terminal derivatizations (R) were introduced in the sequence R-X1KLFKKILKX10L-NH ...

Inhibition of P. expansum infection in apple fruits.

First, the influence of the sequence of peptide application and pathogen inoculation on the inhibition of P. expansum infections in apple fruits was evaluated. The following three protocols were compared: (i) peptide BP76 was preventatively applied to the wound before spore inoculation, (ii) it was mixed with spores and immediately applied, or (iii) it was mixed with spores, preincubated, and subsequently applied (Fig. (Fig.5).5). The results showed that the protocol used had a significant effect on the inhibition of infection. BP76 was highly effective when mixed with spores and preincubated, whereas it was ineffective when applied preventatively. Despite these results, the preventative strategy was chosen because it mimics the treatments used for field control of plant diseases.

FIG. 5.
Effect of the strategy of peptide application and pathogen inoculation on infection by P. expansum in apple wounds. The peptide BP76 was assayed following three protocols: (i) applied to wounds 1 h before pathogen inoculation (preventative, black bars); ...

Next, the most active peptides were tested at 200 and 500 μM using the preventative treatment. The results showed that even though peptides were effective at both concentrations, they were not sufficiently discriminated (data not shown). Therefore, a peptide concentration of 300 μM and a preventative strategy were used in the following assays. Notably, peptides did not produce symptoms of phytotoxicity when applied to apple wounds at the above concentrations.

Finally, the inhibitory activity of the peptides against P. expansum infection was evaluated. Two experiments were performed. All peptides tested (BP21 {Ac,F,V}, BP22 {Ts,F,V}, BP23 {Bz,F,V}, BP29 {Ac,K,V}, BP34 {Ac,L,V}, BP37 {Bn,L,V}, and BP71 {H,Y,F}) significantly decreased apple rot lesion size at 300 μM compared to that of the nontreated control, except for BP71 in the first experiment (Fig. (Fig.6).6). The most active peptide in both experiments was BP22 with an average efficacy of 56% disease reduction. This efficacy was not significantly different than that of a commercial formulation of the reference fungicide imazalil in the first experiment, but it was lower in the second experiment.

FIG. 6.
Effect of the preventative application of peptides in wounds of apple fruits on P. expansum infection. Wounds were treated with peptides at 300 μM and inoculated with conidia, and rot lesion diameter was determined after 11 days of incubation ...

DISCUSSION

Synthetic cecropin A (2-8)-melittin (6-9) hybrid undecapeptides (CECMEL11) with the general structure R-X1KLFKKILKX10L-NH2 were evaluated against Fusarium oxysporum, Penicillium expansum, Aspergillus niger, and Rhizopus stolonifer. Sequences highly active in vitro against F. oxysporum (MIC < 2.5 μM) and P. expansum (MIC < 12.5 μM) were found. To compare the potency of these peptides with other reported antifungal sequences, it has to be taken into account that the type of culture medium influences the MICs. For example, the hexapeptide PAF26 exhibited a MIC of 4 to 6 μM when tested against Penicillium digitatum in 10- to 20-fold-diluted PDB (36-38), whereas the MIC increased to 20 to 40 μM when using undiluted broth (27). Using the latter conditions, other synthetic hexapeptides (26) and the antimicrobial peptide MSI99 (2) showed MICs of 20 to 80 μM against the fungal species P. digitatum, Botrytis cinerea, and F. oxysporum. The best CECMEL11 peptides were highly active against P. expansum in spite of the fact that the assays were performed using undiluted broth. This is in agreement with the reported activity for two of our peptides against P. digitatum (37).

Spores are resting structures of fungi with high survival capacity under adverse conditions and are the most difficult stages to control (43). Therefore, the sporicidal activity of antifungal peptides is critical for efficient control of fungal diseases. Several CECMEL11 antifungal peptides have been found to be sporicidal against F. oxysporum and P. expansum at concentrations around the MIC. Notably, the best peptides killed 99 to 99.9% of the F. oxysporum spores in 35 min at 20 μM. These results correspond to decimal reduction times of 10 to 15 min, which are comparable with the ones reported for currently used antiseptics and disinfectants (30). Moreover, the correlation observed between sporicidal activity and Sytox green uptake suggests that fungal membrane disruption could be the primary mode of action. A similar behavior has been described for other antifungal peptides such as tetralipopeptides (28), the hexapeptide PAF26 (36), and several cecropin-derived peptides (11, 14). However, a correlation between cell permeation and antimicrobial activity is not always observed, because some antimicrobial peptides do not disrupt bacterial membranes or have additional modes of action, including intracellular targets (16, 31, 46, 48).

Antimicrobial peptides derived from natural compounds or de novo designed have been reported to be antibacterial, antifungal, or both (29, 34). The screening of the CECMEL11 library against plant pathogenic bacteria and fungi led to the identification of peptides covering all possibilities. When the library was tested against plant pathogenic bacteria, a set of 15 peptides with high antibacterial activity was identified (4). The evaluation of the library for activity against P. expansum led to the identification of another set of 15 peptides displaying high antifungal activity. The two sets had four peptides in common, with both high antibacterial and antifungal activities. As a matter of example, peptide BP100 was strongly antibacterial but poorly antifungal, BP21 displayed strong antifungal activity but poor antibacterial activity, and BP15 was both antibacterial and antifungal.

Our results confirm previous data on how subtle changes in a peptide sequence influence antimicrobial activity (4, 27, 33). The specificity observed for antimicrobial peptides has been reported to depend on both the peptide and the microorganism tested (41). Among the parameters that modulate peptide activity are the ability to adopt an amphipathic structure, the net positive charge, and the overall hydrophobicity (21). In particular, we previously found that the CECMEL11 peptides with the highest antibacterial activity shared the following structural features: a net charge of +4 to +6, a Lys at position 1, and an aromatic residue at position 10. Moreover, N-terminal derivatization led to less active peptides (4). In contrast, different structural requirements were associated with high antifungal activity against P. expansum. The best peptides bear a charge of +4 or +5 and a valine at position 10 and were N-terminal acetylated. On the other hand, the features of the target membrane that may influence peptide specificity are the structure, the length, and the complexity of the hydrophilic polysaccharide in its outer layer. Based on the results obtained in this study, it is tempting to speculate that subtle lipid composition differences of fungal membranes (or conidial walls) are responsible for the different activity and specificity profiles of the CECMEL11 peptides.

Among the sequences with highest in vitro antifungal activity against P. expansum, peptide Ts-FKLFKKILKVL-NH2 (BP22), when applied preventatively to apple wounds, showed an efficacy slightly lower than that of a commercial fungicide formulation of imazalil. This lower efficacy could be attributed to the inactivation of the peptide and to not being formulated. A certain degree of peptide inactivation by intracellular fluids or cell compounds is expected. In fact, it has been reported that plant protein extracts or intracellular fluids of tomato or tobacco strongly decrease the activity of antimicrobial peptides (11). On the other hand, in many reports, the in vivo tests are performed by inoculating a mixture of fungal spores and the peptide into a wound made into the plant material. In contrast, CECMEL11 peptides were applied into the wound and absorbed by fruit tissues, and subsequently, the pathogen was inoculated into the treated site. Obviously, in the first approach, the peptide has more chance to interact with the target, leading to a dramatic decrease of the number of live cells that are inoculated and, thus, giving disease overcontrol. This could explain that, in our case, a higher peptide concentration (300 μM) was necessary to control apple infections than that reported for peptides inoculated as a mixture with spores (100 to 200 μM) (37). However, a preventative strategy is more realistic because it mimics the standard application of commercial fungicides, such as imazalil, in postharvest rot control. Moreover, it has been described that formulation can greatly improve the antimicrobial activity of pesticides (25). In this study, unlike imazalil, which was used as a commercial formulation, CECMEL11 peptides were applied as aqueous solutions.

Toxicity to animal or plant cells is always a problem with antimicrobial peptides targeted to bacteria or fungi when the mechanism of action is based on the interaction with cell membranes. Even though the hemolytic activity of CECMEL11 peptides was significant at 150 μM, this concentration is around 30 times the MIC, and it is of the same order as that of the reference fungicide imazalil.

In conclusion, the reported technology seems to be feasible because the effective doses of the CECMEL11 peptides are of the same order as those of current fungicides, and despite being unformulated, their level of efficacy is only slightly lower. The major challenge of this research is to meet the requirement of a low production cost for these antimicrobial peptides for food and agricultural use. Molecular farming of these peptides is currently under development and evaluation.

Acknowledgments

R.F. was recipient of a predoctoral fellowship from the Ministry of Education and Science of Spain (MEC). This work was supported by grants AGL2003-03354, AGL2004-07799-C03-01, AGL2006-13564/AGR, and PPT-060000-2008-2 from MICIN of Spain and CIRIT 2005SGR00835 and 2005SGR00275 from the Catalonian government.

Footnotes

[down-pointing small open triangle]Published ahead of print on 17 July 2009.

REFERENCES

1. Agrios, G. N. 1998. Plant pathology, 4th ed. Academic Press, San Diego, CA.
2. Alan, A. R., and E. Earle. 2002. Sensitivity of bacterial and fungal plant pathogens to the lytic peptides, MSI-99, magainin II, and cecropin B. Mol. Plant-Microbe Interact. 15:701-708. [PubMed]
3. Ali, G. S., and A. S. N. Reddy. 2000. Inhibition of fungal and bacterial plant pathogens by synthetic peptides: in vitro growth inhibition, interaction between peptides, and inhibition of disease progression. Mol. Plant-Microbe Interact. 13:847-859. [PubMed]
4. Badosa, E., R. Ferre, M. Planas, L. Feliu, E. Besalú, J. Cabrefiga, E. Bardají, and E. Montesinos. 2007. A library of linear undecapeptides with bactericidal activity against phytopathogenic bacteria. Peptides 28:2276-2285. [PubMed]
5. Bardají, E., E. Montesinos, E. Badosa, L. Feliu, M. Planas, and R. Ferre. April 2006. Antimicrobial linear peptides. Patent WO/2007/125142 A1.
6. Bonaterra, A., M. Mari, L. Casallini, and E. Montesinos. 2003. Biological control of Monilinia laxa and Rhizopus stolonifer in postharvest of stone fruit by Pantoea agglomerans EPS125 and putative mechanisms of antagonism. Int. J. Food Microbiol. 84:93-104. [PubMed]
7. Broekaert, W. F., B. P. A. Cammue, M. F. C. DeBolle, K. Thevissen, G. W. De Samblanx, and R. W. Osborn. 1997. Antimicrobial peptides from plants. Crit. Rev. Plant Sci. 16:297-323.
8. Brogden, K. A. 2005. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol. 3:238-250. [PubMed]
9. Brogden, K. A., M. Ackermann, P. B. McCray, Jr., and B. F. Tack. 2003. Antimicrobial peptides in animals and their role in host defences. Int. J. Antimicrob. Agents 22:465-478. [PubMed]
10. Bulet, P., R. Stöcklin, and L. Menin. 2004. Antimicrobial peptides: from invertebrates to vertebrates. Immunol. Rev. 198:169-184. [PubMed]
11. Cavallarin, L., D. Andreu, and B. San Segundo. 1998. Cecropin A-derived peptides are potent inhibitors of fungal plant pathogens. Mol. Plant-Microbe Interact. 11:218-227. [PubMed]
12. De Lucca, A. J., and T. J. Walsh. 1999. Antifungal peptides: novel therapeutic compounds against emerging pathogens. Antimicrob. Agents Chemother. 43:1-11. [PMC free article] [PubMed]
13. De Lucca, A. J., and T. J. Walsh. 2000. Antifungal peptides: origin, activity and therapeutic potential. Rev. Iberoam. Micol. 17:116-120. [PubMed]
14. DeLucca, A. J., J. M. Bland, T. J. Jacks, C. Grimm, T. E. Cleveland, and T. J. Walsh. 1997. Fungicidal activity of cecropin A. Antimicrob. Agents Chemother. 41:481-483. [PMC free article] [PubMed]
15. Deshayes, S., M. C. Morris, G. Divita, and F. Heitz. 2005. Cell-penetrating peptides: tools for intracellular delivery of therapeutics. Cell. Mol. Life Sci. 62:1839-1849. [PubMed]
16. Epand, R. M., and H. J. Vogel. 1999. Diversity of antimicrobial peptides and their mechanisms of action. Biochim. Biophys. Acta 1462:11-28. [PubMed]
17. Ferre, R., E. Badosa, L. Feliu, M. Planas, E. Montesinos, and E. Bardají. 2006. Inhibition of plant-pathogenic bacteria by short synthetic cecropin A-melittin hybrid peptides. Appl. Environ. Microbiol. 72:3302-3308. [PMC free article] [PubMed]
18. Francés, J., A. Bonaterra, M. C. Moreno, J. Cabrefiga, E. Badosa, and E. Montesinos. 2006. Pathogen aggressiveness and postharvest biocontrol efficiency in Pantoea agglomerans. Postharvest Biol. Technol. 39:299-307.
19. Ganz, T., and R. I. Lehrer. 1998. Antimicrobial peptides of vertebrates. Curr. Opin. Immunol. 10:41-44. [PubMed]
20. García-Olmedo, F., A. Molina, J. M. Alamillo, and P. Rodríguez-Palenzuela. 1998. Plant defense peptides. Biopolymers 47:479-491. [PubMed]
21. Giangaspero, A., L. Sandri, and A. Tossi. 2001. Amphipathic alpha-helical antimicrobial peptides: a systematic study of the effects of structural and physical properties on biological activity. Eur. J. Biochem. 268:5589-5600. [PubMed]
22. Hernández-Lauzardo, A. N., S. Baustista-Baños, M. G. Velázquez-del Valle, M. G. Méndez-Montealvo, M. M. Sánchez-Rivera, and L. A. Bello-Pérez. 2008. Antifungal effects of chitosan with molecular weights on in vitro development of Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. Carbohydr. Polym. 73:541-547.
23. Jenssen, H., P. Hamill, and R. E. W. Hancock. 2006. Peptide antimicrobial agents. Clin. Microbiol. Rev. 19:491-511. [PMC free article] [PubMed]
24. Kamysz, W., A. Krolicka, K. Bogucka, T. Ossowski, J. Lukasiak, and E. Lojkowska. 2005. Antibacterial activity of synthetic peptides against plant pathogenic Pectobacterium species. J. Phytopathol. 153:313-317.
25. Knowles, D. A. 1998. Chemistry and technology of agricultural formulations. Kluwer Academic, London, United Kingdom.
26. López-García, B., L. González-Candelas, E. Pérez-Payá, and J. F. Marcos. 2000. Identification and characterization of a hexapeptide with activity against phytopathogenic fungi that cause postharvest decay in fruits. Mol. Plant-Microbe Interact. 13:837-846. [PubMed]
27. López-García, B., E. Pérez-Payá, and J. F. Marcos. 2002. Identification of novel hexapeptides bioactive against phytopathogenic fungi through screening of a synthetic peptide combinatorial library. Appl. Environ. Microbiol. 68:2453-2460. [PMC free article] [PubMed]
28. Makovitzki, A., D. Avrahami, and Y. Shai. 2006. Ultrashort antibacterial and antifungal lipopeptides. Proc. Natl. Acad. Sci. USA 103:15997-16002. [PMC free article] [PubMed]
29. Marcos, J., A. Muñoz, E. Pérez-Payá, S. Misra, and B. López-García. 2008. Identification and rational design of novel antimicrobial peptides for plant protection. Annu. Rev. Phytopathol. 46:271-301. [PubMed]
30. Mazzola, P. G., T. C. Vessoni, and A. M. Martins. 2003. Determination of decimal reduction time (D value) of chemical agents used in hospitals for disinfection purposes. BMC Infect. Dis. 3:24. [PMC free article] [PubMed]
31. McPhee, J. B., M. G. Scott, and R. E. W. Hancock. 2005. Design of host defence peptides for antimicrobial and immunity enhancing activities. Comb. Chem. High Throughput Screen. 8:257-272. [PubMed]
32. Monroc, S., E. Badosa, L. Feliu, M. Planas, E. Montesinos, and E. Bardají. 2006. De novo designed cyclic peptides as inhibitors of plant pathogenic bacteria. Peptides 27:2567-2574. [PubMed]
33. Monroc, S., E. Badosa, E. Besalú, M. Planas, E. Bardají, E. Montesinos, and L. Feliu. 2006. Improvement of cyclic decapeptides against plant pathogenic bacteria using a combinatorial chemistry approach. Peptides 27:2575-2584. [PubMed]
34. Montesinos, E. 2007. Antimicrobial peptides and plant disease control. FEMS Microbiol. Lett. 270:1-11. [PubMed]
35. Montesinos, E., and E. Bardají. 2008. Synthetic antimicrobial peptides as agricultural pesticides for plant-disease control. Chem. Biodivers. 5:1225-1237. [PubMed]
36. Muñoz, A., B. López-Garcia, and J. F. Marcos. 2006. Studies on the mode of action of the antifungal hexapeptide PAF 26. Antimicrob. Agents Chemother. 50:3847-3855. [PMC free article] [PubMed]
37. Muñoz, A., B. López-García, and J. F. Marcos. 2007. Comparative study of antimicrobial peptides to control citrus postharvest decay caused by Penicillium digitatum. J. Agr. Food Chem. 55:8170-8176. [PubMed]
38. Muñoz, A., and J. F. Marcos. 2006. Activity and mode of action against fungal phytopathogens of bovine lactoferricin-derived peptides. J. Appl. Bacteriol. 101:1-9. [PubMed]
39. Ogawa, J. M., E. I. Zehr, G. W. Bird, D. F. Ritchie, K. Uriu, and J. K. Uyemoto (ed.). 1995. Compendium of stone fruit diseases. APS Press, St. Paul, MN.
40. Otvos, L., Jr. 2000. Antibacterial peptides isolated from insects. J. Pept. Sci. 6:497-511. [PubMed]
41. Papo, N., and Y. Shai. 2003. Exploring peptide membrane interaction using surface plasmon resonance: differentiation between pore formation versus membrane disruption by lytic peptides. Biochemistry 42:458-466. [PubMed]
42. Powell, W. A., C. M. Catranis, and C. A. Maynard. 1995. Synthetic antimicrobial peptide design. Mol. Plant-Microbe Interact. 8:792-794. [PubMed]
43. Russell, A. D., W. D. Hugo, and G. A. J. Ayliffe (ed.). 1982. Principles and practice of disinfection, preservation and sterilization. Blackwell Science, Oxford, United Kingdom.
44. Scheffknecht, S., R. Mammerler, S. Steinkellner, and H. Vierheilig. 2006. Root exudates of mycorrhizal tomato plants exhibit a different effect on microconidia germination of Fusarium oxysporum f. sp. lycopersici than root exudates from nonmycorrhizal tomato plants. Mycorrhiza 6:365-370. [PubMed]
45. Sholberg, P. L., and W. S. Conway. 2004. Postharvest pathology. In K. C. Gross, C. Y. Wang, and M. Saltveit (ed.), Agriculture handbook number 66: the commercial storage of fruits, vegetables, and florist and nursery stocks. U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS), Washington, DC. http://www.ba.ars.usda.gov/hb66/contents.html.
46. Steffen, H., S. Rieg, I. Wiedemann, H. Kalbacher, A. Deeg, H. G. Sahl, A. Peschel, F. Götz, C. Garbe, and B. Schittek. 2006. Naturally processed dermcidin-derived peptides do not permeabilize bacterial membranes and kill microorganisms irrespective of their charge. Antimicrob. Agents Chemother. 50:2608-2620. [PMC free article] [PubMed]
47. Weidenbörner, M. 2001. Encyclopedia of food mycotoxins. Springer, Berlin, Germany.
48. Yount, N. Y., and M. R. Yeaman. 2005. Immunocontinuum: perspectives in antimicrobial peptide mechanisms of action and resistance. Protein Pept. Lett. 12:49-67. [PubMed]
49. Zasloff, M. 2002. Antimicrobial peptides of multicellular organisms. Nature 415:389-395. [PubMed]

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