Int. J. Mol. Sci. 2012, 13, 15042-15053; doi:10.

3390/ijms131115042
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International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
Article

Antifungal Activity of (KW)n or (RW)n Peptide against

Fusarium solani and Fusarium oxysporum
Ramamourthy Gopal 1, Hyungjong Na 2, Chang Ho Seo 3 and Yoonkyung Park 1,2,*
1
Research Center for Proteineous Materials, Chosun University, Gwangju 501-759, Korea;
E-Mail: [email protected]
2
Department of Biotechnology, Chosun University, Gwangju 501-759, Korea;
E-Mail: [email protected]
3
Department of Bioinformatics, Kongju National University, Kongju 314-701, Korea;
E-Mail: [email protected]

* Author to whom correspondence should be addressed; E-Mail: [email protected];

Tel.: +82-62-230-6854; Fax: +82-62-225-6758.

Received: 16 July 2012; in revised form: 14 August 2012 / Accepted: 17 October 2012 /
Published: 15 November 2012

Abstract: The presence of lysine (Lys) or arginine (Arg) and tryptophan (Trp) are important
for the antimicrobial effects of cationic peptides. Therefore, we designed and synthesized a
series of antimicrobial peptides with various numbers of Lys (or Arg) and Trp repeats [(KW
and RW)n-NH2, where n equals 2, 3, 4, or 5]. Antifungal activities of these peptides
increased with chain length. Light microscopy demonstrated that longer peptides (n = 4, 5)
strongly inhibited in vitro growth of Fusarium solani, and Fusarium oxysporum, at
4–32 μM. Furthermore, longer peptides displayed potent fungicidal activities against a
variety of agronomical important filamentous fungi, including F. solani and F. oxysporum,
at their minimal inhibitory concentrations (MICs). However, RW series peptides showed
slightly higher fungicidal activities than KW peptides against the two strains. Taken
together, the results of this study indicate that these short peptides would be good candidates
for use as synthetic or transgenic antifungal agents.

Keywords: lysine; arginine; tryptophan; antifungal peptides; fungicidal

Int. J. Mol. Sci. 2012, 13 15043

1. Introduction

Cationic antifungal peptides have been developed as novel biocidal agents in the battle against
pathogenic microorganisms [1]. Generally, cationic peptides are 12 to 50 amino acids in length as well
as amphiphilic, as they contain two to nine basic residues (arginine (Arg) or lysine (Lys)) and
approximately 50% hydrophobic residues [2,3]. However, the large size of antimicrobial peptides
(AMPs) hinders their use due to high manufacturing costs. Therefore, selective short AMPs have been
developed based on their amino acid combinations, charge, and hydrophobicity [4–8]. In this context,
AMPs containing cationic and hydrophobic amino acids constitute a promising tool to combat plant
fungal pathogens. For example, both defensin and MtDef4 require cationic and hydrophobic amino acids
for their antifungal activities [9]. Furthermore, this requirement has been demonstrated by the loss of
antimicrobial activity upon substitution of basic residues in AMPs [10]. Other short tryptophan
(Trp)-rich cationic peptides, PAF26, PAF38, PAF40, and BM0, also display antifungal activity [11,12].
The peptide indolicidin, which belongs to the cathelicidin family of AMPs, is an Arg and Trp-rich
peptide with potent, broad-spectrum antimicrobial activity [13,14]. Further, a large number of cationic
plant defensins exhibit inhibitory activities against filamentous fungi both in vitro and in transgenic
plants [15–20]. It has been reported that short peptides show antifungal activity as they contain cationic
and hydrophobic amino acids [21,22]. This is clearly evident based on previous reports that
hexapeptides containing predominately cationic and hydrophobic amino acids show the highest
antifungal activity [11,23]. Therefore, we synthesized a series of peptides containing a repeated pattern
of Lys (K) or Arg (R) and Trp (W) residues, (KW)n and (RW)n (where n equals 2, 3, 4, or 5) (Figure 1),
and determined their antifungal and fungicidal activities.

Figure 1. Chemical structure of linear antimicrobial peptides (AMPs) (KW)n-NH2 or

(RW)n-NH2 used in this study, where n = 2, 3, 4, and 5.

2. Results and Discussion

Antifungal peptides have been used in both transgenic plant and human pharmaceutical applications.
Furthermore, it has been extensively reported that many short AMPs rich in Lys or Arg and Trp show
antifungal activity [4,6,8,24–26]. Although it is not yet clear that these peptides kill plant fungal
pathogens, it is important to optimize chain length of AMPs for the purpose of large-scale production. In
the present study, we investigated the antimicrobial effects of small KW and RW series peptides against
plant pathogens such as Fusarium solani and Fusarium oxysporum. Specifically, F. solani is a
Int. J. Mol. Sci. 2012, 13 15044

phytopathogenic fungus as well as an important causal agent of several crop diseases, including root and
stem rot of pea, sudden death syndrome of soybean, foot rot of bean, and dry rot of potato [27–30].
F. oxysporum is a phytopathogenic fungus that affects tomato crops, causing huge losses to farmers [31].

2.1. Antifungal Activities against Hyphal Growth

In the present study, spectrophotometric and microscopic experiments were used to assess the
antifungal activities of the peptides on hyphal growth of F. solani and F. oxysporum. As shown in Figure 2,
chain length of (KW)n and (RW)n peptides strongly correlated with increasing antifungal activity.
Decamer (KW and RW)5 peptides inhibited conidial germination completely (100% growth inhibition)
at 4 μM for F. solani and 8 μM for F. oxysporum. Further, decamer peptides reduced growth rates
compared to other peptides at all tested concentrations (1, 2, 4, 8, 16, 32, and 64 μM), although a
concentration of 1 μM did not prevent sporulation. Similarly, (KW)4 and (RW)4 peptides exhibited
antifungal effects against F. solani at concentrations as low as 4 μM, which is two-fold less than their
minimal inhibitory concentration (MIC) (8 μM). (KW)4 and (RW)4 peptides also inhibited growth of
F. oxysporum at concentrations below their MIC (16 μM). Specifically, at a concentration of 8 μM,
(KW)4 and (RW)4 inhibited growth of F. oxysporum by 45% and 55%, respectively (Figure 2C,D).
(KW)3 and (RW)3 peptides at a concentration of 16 μM completely inhibited germination of F. solani
conidia. In contrast, F. oxysporum conidia were able to germinate and grow even in the presence of
peptides at 16 μM, although 100% growth inhibition was observed at 32 μM (Figure 2C,D). Thus,
(KW)5 and (RW)5 peptides exhibited significantly higher antifungal activities than other peptides and
were almost as potent as melittin. Lastly, (KW)2 and (RW)2 at 64 μM did not inhibit growth of the fungal
strains. The antifungal activities of peptides were in the following order: tetrameric peptides <
hexameric peptides < octameric peptides < decameric peptides (Figure 2). These data suggest that a
critical chain length may be required for significant antifungal activity. Melittin possesses strong
antifungal activity and served as a positive control in this experiment. Photomicrographs of the mycelia
of F. solani and F. oxysporum fungi were taken after a 24 h growth period. The effects of peptides on
hyphal morphology were monitored and compared with the morphology of untreated hyphae
(Figure 3A). Decameric and octameric peptides significantly inhibited spore germination and hyphal
growth of phytopathogenic fungi in comparison with tetrameric peptides, which is consistent with the
results on percentage inhibition of fungal growth. However, above 16 μM (for F. solani) or 32 μM (for
F. oxysporum), (KW)3 and (RW)3 peptides noticeably inhibited conidial germination and hyphal
development (Figure 3B). Significantly, hyberbranching of fungal hyphae, which is a typical
morphological response of F. solani and F. oxysporum in response to (KW)2 and (RW)2, was not
observed in the presence of other peptides at concentrations above or near their MICs. Therefore, we
believe that antifungal activity increased with chain length. A previous study showed that (RW)n series
peptides with increased chain length possess enhanced antimicrobial activity [6]. Furthermore,
decameric peptides and melittin showed similar antifungal activities against F. solani and F. oxysporum.
On the other hand, hexametic and octameric peptides showed decreased antifungal activities against the
same two strains in comparison with melittin. This result is consistent with previous data that also
indicated that small cationic hexapeptides had lower antimicrobial activities compared to longer,
naturally occurring AMPs such as magainin, cecropin, and melittin [11,32]. In addition, these hexameric
Int. J. Mol. Sci. 2012, 13 15045

peptides showed higher antifungal activities than the other hexameric peptides [21], suggesting that, in
this (KW)3 or (RW)3, the composition of amino acid within the repeating motif seems to be more
selective than the PAF26 or PAF32 for fungal membrane.

Figure 2. Quantitative measurement of fungal growth inhibition. (KW)n-NH2 (A and C) and

(RW)n-NH2 (B and D). Antimicrobial activities of tetrapeptides (squares), hexapeptides
(triangle), octapeptides (circle), decapeptides (diamonds), and melittin (cross) aganist
in vitro growth of Fusarium solani (A and B) and Fusarium oxysporum (C and D).

Figure 3. (A) Images showing non-inhibition of conidial germination and hyphal growth of
fungal strains treated with (KW)2-NH2 or (RW)2-NH2 at 64 μM. Images showing inhibition
of conidial germination and hyphal growth of Fusarium solani (B) and Fusarium oxysporum
(C) at different concentrations of peptides. Bar = 50 μm.
Int. J. Mol. Sci. 2012, 13 15046

Figure 3. Cont.
Int. J. Mol. Sci. 2012, 13 15047

Figure 3. Cont.
Int. J. Mol. Sci. 2012, 13 15048

2.2. Effects of Longer Peptides on Cell Viability

To determine whether the antifungal peptides are fungicidal or fungistatic, F. solani and
F. oxysporum cells were treated with or without peptides for 3 h. (KW)3 and (RW)3 peptides at their
MICs showed moderate fungicidal activities against both F. solani and F. oxysporum. After treatment
with (KW)4 and (RW)4 at their MICs, 68% and 69% of F. solani (or 74% and 76% of F. oxysporum)
cells were killed, respectively (Figure 4). For (KW5) and (RW5) at their MICs, 72% and 75% of
F. solani cells along with 76% and 79% of F. oxysporum cells were killed, respectively. The percentages
for F. oxysporum further increased to 89% and 92% in the presence of (KW)5 and (RW)5 at 32 μM,
respectively. Consistent with the results on percentage of growth inhibition, killing activity increased
with peptide concentration as well as peptide length. Moreover, the results show that both (KW)n and
(RW)n (n = 3, 4, and 5) had similar inhibitory activities against the two strains at corresponding MICs,
but they differed in their fungicidal activities. In fact, RW series peptides showed slightly higher
fungicidal activity than KW peptides. Some studies also reported that Arg-containing peptides were
more active against plant fungal strains than Lys-containing peptides [20]. Specifically, the guanidinium
group of Arg strongly interacts with fungal strains due to its higher net positive charge compared to the
protonated amine of lysine. In addition, Arg-containing peptides showed a two-fold greater activity
against F. solani than F. oxysporum. Taken together, our results clearly indicate that the level of
antimicrobial activity depends on the peptide sequence and fungal membrane composition.

Figure 4. Viability of hyphal cells after treatment with antifungal peptides. Viability of
fungal cells after treatment with peptides at different concentrations was monitored using an
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based assay.

Our results also show that the peptides act as fungicidal compounds. Application of fungicidal agents
is usual practice when fighting plant diseases [33]. Specifically, antifungal peptides with fungicidal
activity are often used in plant transgenic applications. For example, antifungal plant defensin is induced
in radish leaves upon challenge with fungal pathogens, suggesting a role in plant defense [34]. Further,
the natural AMP magainin has been expressed in Nicotiana tabacum to develop resistance to
phytopathogens [35]. Other studies also reported that transgenic plants expressing AMPs exhibit
broad-spectrum resistance to phytopathogen infection [36–38]. At present, the high cost of synthetic
Int. J. Mol. Sci. 2012, 13 15049

peptides constitutes an obvious limitation to agricultural and food applications. As a result, there have
been many efforts to express short AMPs in transgenic plants [38,39], for example indolicidin [40,41].
Short hexapeptides are used to control postharvest diseases in fruits and vegetables caused by fungal
phytopathogens [21,23,42]. In fact, we previously identified (KW)4 as a potentially non-toxic AMP [43]
that can be produced on a large scale economically, although this short peptide has been produced in
transgenic plants. Future research will determine the feasibility of these options. Meanwhile, the modes
of action of these peptides are yet to be resolved, although studies have proposed various killing
mechanisms for Lys-Arg/Trp rich AMPs, including cytoplasmic membrane disruption and inhibition of
nucleic acid synthesis [44]. Other studies have reported that Lys-Arg/Trp-rich AMPs kill fungal strains
through nucleic acid binding or cell penetration [45,46]. Studies on the modes of action of these peptides
are currently underway.

3. Experimental Section

3.1. Materials

Rink amide 4-methylbenzhydrylamine resin, fluoren-9-ylmethoxycarbonyl (Fmoc) amino acids, and

other reagents for peptide synthesis were purchased from Calibiochem-Novabiochem (La Jolla, CA,
USA). For the quantitative antifungal assay and fungicidal activity assay, the following fungal strains
were obtained from the Korea Collection for Type Cultures (KCTC): F. solani (KCTC 6326) and
F. oxysporum (KCTC 6076). Fungal cells were grown on PDA (potato dextrose agar) plate and
subcultured for 2–3 weeks.

3.2. Peptide Synthesis and Purifications

The peptides KWKW-NH2 (KW)2, KWKWKW-NH2 (KW)3, KWKWKWKW-NH2 (KW)4,

KWKWKWKWKW-NH2 (KW)5, RWRW-NH2 (RW)2, RWRWRW-NH2 (RW)3, RWRWRWRW-NH2
(RW)4, RWRWRWRWRW-NH2 (RW)5 and GIGAVLKVLTTGLPALISWIKRKRQQ (melittin) were
synthesized by the solid-phase method using Fmoc chemistry on a solid support of rink amide
4-methylbenzhydrydrylamine resin. Then, 0.1 M N-hydroxy benzotriazole (HOBt) and 0.45 M
2-(1H-benzotriazole-1-yil)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) in dimethylformamide
(DMF) along with 2 M N,N-diisopropyl ethylamine (DIEA) in N-methylpyrrolidone (NMP) were used
as coupling reagents, and 10-fold excess Fmoc-amino acid was added during every coupling cycle.
Following a final deprotection with a solution of 20% piperidine in DMF and cleavage with a mixture of
TFA/water/triisopropylsilane (90:5:5) for 2 h at room temperature [47], the crude peptides were
repeatedly extracted with diethyl ether and purified using reverse phase preparative high performance
liquid chromatography (HPLC) on a Vydac C18 column (4.6 × 250 mm, 300 Å, 5 nm). The molecular
masses of the peptides were confirmed by using a matrix-assisted laser desorption ionization mass
spectrometer (data not shown) (MALDI II, Kratos Analytical Ins.). The purity of all peptides were found
to be > 95%.
Int. J. Mol. Sci. 2012, 13 15050

3.3. Computational Modeling

Chemical structures of the peptides were built using ChemOffice Desktop 2004 for Windows
(CambridgeSoft (CS) Corporation, Cambridge, MA, USA).

3.4. Antifungal Assay

Fungal fragments, precultured in mycelial growth medium, were placed in the center of PDA plates,
after which the cultures were incubated for 96 h at 25 °C in the dark. After incubation, spores were
isolated from cultures growing in half-strength PDA. Spore concentrations were then adjusted to
5 × 104 spores/mL in half-strength PDA, after which 80 μL was added to the wells of sterile 96-well
flat-bottomed microtiter plates along with 20 μL of peptide or media to give final concentrations of
1–64 μM. Several wells were kept untreated as a control to monitor fungal growth. Plates were incubated
in the dark at 25 °C for 24 h before hyphal growth was determined by measuring optical density at
595 nm using a microtiter plate Elisa reader (Molecular Devices, Sunnyvale, CA, USA) [48]. Each test
was performed in triplicate. Percentages of inhibition were then calculated (Figure 1) (0% inhibition
indicates growth equal to control sample (only media)) [21]. The lowest concentration of peptide
inhibiting fungal growth was monitored microscopically with an inverted light microscope (IX71,
Olympus, Tokyo, Japan) [49].

3.5. Cell Viability Assay

The spore suspension at a concentration of 5 × 104 spores/mL (80 μL) was transferred to a 96-well
microtiter plate along with 20 μL of (KW)n or (RW)n peptides (n = 3, 4, and 5), melittin, or media to give
final peptide concentrations of 4–32 μM. Plates were incubated for 3 h before the addition of
10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 5 mg/mL; Sigma).
Plates were then incubated again for 16 h at room temperature, followed by the addition of 100 μL of
MTT solvent (0.1 N HCl in anhydrous isopropyl alcohol). Presence of MTT/formazan was monitored
spectrophotometrically by measuring the absorbance at 570 nm and then subtracting the background
absorbance at 690 nm using a Versa-Max microplate Elisa reader (Molecular Devices, Sunnyvale, CA,
USA) [48]. Each measurement was conducted in triplicate.

4. Conclusions

In summary, increased chain length along with a higher ratio between hydrophobicity and net charge
resulted in increased antifungal and fungicidal activities. Our results confirm that KW and RW peptide
elements could be incorporated into the development of an AMP with low cost due to their short lengths,
making these peptides a promising alternative tool in the field of green biocides.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the
Korea government (MEST) (No. 2011-0017532) and the Human Resource Training Project for
Regional Innovation.
Int. J. Mol. Sci. 2012, 13 15051

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