background image

J. Microbiol. Biotechnol

. (2009),

 

19

(8), 760–764

doi: 10.4014/jmb.0812.649
First published online 28 April 2009

An 

In Vitro

 Study of the Antifungal Effect of Silver Nanoparticles on Oak

Wilt Pathogen 

Raffaelea

 sp.

Kim, Sang Woo

1

, Kyoung Su Kim

1

, Kabir Lamsal

1

, Young-Jae Kim

1

, Seung Bin Kim

2

, Mooyoung Jung

3

,

Sang-Jun Sim

4

, Ha-Sun Kim

4

, Seok-Joon Chang

4

, Jong Kuk Kim

5

, and Youn Su Lee

1

*

1

Division of Bio-Resources Technology, Kangwon National University, Chuncheon 200-701, Korea

2

Department of Chemistry, POSTECH, Pohang 790-784, Korea

3

School of Technology Management, Ulsan National Institute of Science and Technology, Ulsan 689-805, Korea

4

Forest Research Institute of Gangwon Province, Chuncheon 200-140, Korea

5

Division of Forest Resources, Kangwon National University, Chuncheon 200-701, Korea

Received: December 2, 2008 / Revised: January 14, 2009 / Accepted: January 23, 2009

In this study, we investigated the antifungal activity of
three different forms of silver nanoparticles against the
unidentified ambrosia fungus 

Raffaelea

 sp., which has

been responsible for the mortality of a large number of
oak trees in Korea. Growth of fungi in the presence of
silver nanoparticles was significantly inhibited in a dose-
dependent manner. We also assessed the effectiveness of
combining the different forms of nanoparticles. Microscopic
observation revealed that silver nanoparticles caused
detrimental effects not only on fungal hyphae but also on
conidial germination. 

Keywords:

 Silver nanoparticles, 

Raffaelea

, ambrosia, oak wilt.

Mortality of oaks in Korea has been rapidly increasing
since 2004. Tens of thousands of oaks have been killed by
an unidentified fungal species of the genus 

Raffaelea. 

Oak

wilt caused by 

Raffaelea

 sp. has become a major destructive

disease in Korea, damaging both forest and landscape
oaks.

 Raffaelea 

sp. is an ambrosia fungus that invades

water-conducting tissues of diverse tree species. This
invasion leads to cavitation, discoloration, desiccation, and
dysfunction of xylem vessels, thereby blocking upward
water flow [12]. Extensive disease development in the
vessels causes severe wilt of foliage and subsequent death
of the host. Ambrosia fungi have a symbiotic relationship
with ambrosia beetles. The fungi serve as a nutrient source
during insect development in galleries and are dispersed to
new habitats primarily 

via

 the mycangium, a specialized

insect organ adapted to the transport of symbiotic fungi
[1, 8, 10]. In Korea, 

Raffaelea

 sp. is predominantly observed

in the galleries and mycangia of an ambrosia beetle,

Platypus koryoensis, 

in wilted oak trees, suggesting that

this fungus is responsible for the oak wilt disease [1, 8, 10].
Similarly, this hypothesis is supported by the results of an
inoculation test in which oak wilt disease in Japan was
proved to be associated with a related fungus, 

R. quercivora

[11]. Identification of the 

Raffaelea 

species that is causing

the death of Korean oaks is a very important task, but it has
yet to be accomplished. Approximately 13 species of the
genus have been reported, including 

R. montetyi, R.

ambrosiae, 

and

 R. tritirachium 

[9]. However, the identification

and classification of species of 

Raffaelea

 have remained

challenging tasks since the early taxonomic establishment
of the genus

 

[6]. This difficulty is due to the lack of distinct

differences in morphological characteristics between species.
For example, conidia and conidiophores lack pigmentation
and other distinguishing features, and conidiogenesis is
barely visible. 

It is very difficult to control oak wilt because the

advanced disease becomes well-established within trees
prior to the appearance of wilt symptoms in summer [12].
Few fungicides have been effective in curing diseased
trees. Use of the systemic fungicide propiconazole (sold
under the trade name Alamo) for oak wilt diseases caused
by the fungal pathogen 

Ceratocystis fagacearum 

is limited

owing to high cost. Current control measures are concentrated
mostly on preventing dissemination of the disease to
uninfected plants; diseased trees are removed and treated
with fumigants. Finding or developing a new fungicide
that is effective against deadly oak wilt pathogens is a
priority for protecting oak species.

*Corresponding author

Phone: +82-33-250-6417; Fax: +82-33-244-6410;
E-mail: younslee@kangwon.ac.kr

background image

761

Kim 

et al.

Silver has been used in many applications in pure free

metal or compound form because it possesses antimicrobial
activity against pathogens but is nontoxic to humans
[3, 18]. Silver ions are very reactive, leading to inhibition of
microbial respiration and metabolism as well as physical
damage [2, 17]. Moreover, it has been suggested that silver
ions intercalate into bacterial DNA once entering the cell,
which prevents further proliferation of the pathogen [4].
Recently, nanotechnology has amplified the effectiveness of
silver particles as antimicrobial agents. The larger surface
area-to-volume ratio of silver nanoparticles increases their
contact with microbes and their abilility to permeate cells.
Nanoparticle development has restored interest in the
antimicrobial effects of metals, which declined following
the widespread application of modern synthetic antibiotics.
Unfortunately, studies on the antimicrobial activity of
silver nanoparticles have been performed mostly on animal
pathogens [2, 3, 4, 15]. In this study, we investigated the
effects of three different forms of silver nanoparticles on
the ascomycetous phytopathogen 

Raffaelea

 sp. that causes

oak wilt in Korea.

M

ATERIALS

 

AND

 M

ETHODS

Fungal Pathogen, Culture Conditions, and Silver Nanoparticles

Raffaelea

 sp. was routinely grown on several media, including 2%

malt agar (MA, 0.2% [w/v] malt extract, 1.5% [w/v] agar) medium
at room temperature. For measurement of hyphal growth, an agar
plug (4 mm in diameter) was obtained from the actively growing
edge of the fungus, inoculated into the center of a culture plate
containing MA medium, and incubated for 7 days at 24

o

C. Silver

nanoparticles (Nanover) were obtained from BioPlus Co., Ltd. (Korea).
The three different forms of Nanover, CV-WA13 (CV), AT-WB13R
(AT), and PR-WB13 (PR), all dissolved in distilled water, were
utilized in this study. These nanoparticles have an average size of
4

-

8 nm.

Conidial Germination

Conidia were obtained from hyphal mats of 

Raffaelea

 sp. grown for

2 weeks on MA medium. Ten ml of sterile distilled water was
added to culture plates, and then conidia were harvested with a glass
rod and filtered through Miracloth (Calbiochem, La Jolla, CA,
U.S.A.). After adjusting the concentration of conidia to 10

4

/ml, 1 ml

of the suspension was spread on MA plates supplemented with either
10 ppm AT or an equal volume of water. The plates were incubated
for 2 days at 24

o

C and used to observe conidial germination. 

Scanning Electron Microscopy (SEM)

A culture of 

Raffaelea

 sp. grown on MA medium plates was

sprayed with 5 ml of AT solution (10 ppm) and incubated for 3
days. This specimen was fixed in 4% glutaraldehyde for 3 h and
treated with 0.1 M cacodylate buffer for 1 h. After washing with
distilled water, the specimen was dehydrated in a graded ethanol
series up to 100%, critical-point dried, and gold-coated using an ion
sputter coater. The specimen was observed under a Hitachi S-3500N
scanning electron microscope at an accelerating voltage of 10 kV. 

R

ESULTS

Effect of Silver Nanoparticles on Growth of 

Raffaelea

 sp.

First, we compared the growth of 

Raffaelea

 sp. on several

synthetic media, including MA, potato dextrose agar (PDA),
DYPA (20 g dextrose, 5 g yeast extract, 2 g peptone, 1.5%
agar), and OA (17 g oatmeal, 1.5% agar). Since the fungus
grew very slowly on commonly used PDA compared with
the other media tested (data not shown), MA medium was
finally selected for routine culture. To evaluate whether silver

Fig. 1.

 Effect of silver nanoparticles on hyphal growth in

Raffaelea

 sp. 

A

. Radial hyphal growth on MA medium containing the indicated

concentrations of each form of silver nanoparticles. Non-treatment served
as a control (top left panel). An agar plug (4 mm in diameter) obtained
from the actively growing edge of the wild-type strain was inoculated in
the center of the plates. Pictures shown were taken at 7 days post-
inoculation. Three independent experiments were performed. 

B

.

 

Relative

hyphal growth rate on MA medium containing silver nanoparticles.
Colony diameters were measured at 7 days post-inoculation. Data were
obtained from triplicate assays; data are presented as means

±

SD.

background image

E

FFECT

 

OF

 S

ILVER

 N

ANOPARTICLES

 

ON

 

A

 F

UNGAL

 P

HYTOPATHOGEN

R

AFFAELEA

 

SP

.

762

nanoparticles possess antifungal activity against 

Raffaelea

sp., the fungus was grown on MA plates supplemented with
various concentrations of each different Nanover form.
Significant inhibition of hyphal growth and abnormal
patches of aerial hyphal mass were observed following
treatment with CV, AT, or PR at concentrations higher than
10 ppm (Figs. 1A and 1B). Measurement of radial hyphal
growth revealed that each type of silver nanoparticles
retarded fungal growth in a dose-dependent manner; the
hyphal growth rate was 0.24, 0.12, or 0.12 at 25 ppm CV,
AT, or PR, respectively, relative to the value of 1 corresponding
to non-treatment (Fig. 1B). Differences in antimicrobial
efficiency among the different forms of silver nanoparticles
were also observed. The inhibition efficiency of CV
overall was less than that of AT and PR. For example, CV
did not inhibit fungal growth at 5 ppm, whereas AT and PR
were effective at this concentration.

Effect of Combining Silver Nanparticles on Fungal
Growth

Since there was a difference in antifungal activity between
the three forms of silver nanoparticles, we investigated the
effect of combining the different forms on fungal growth.
As shown in Fig. 2, when CV was combined with either
AT or RP, increased inhibition of fungal growth was clearly
observed (Fig. 2A), compared with treatment with CV
alone. In particular, we observed relative fungal growth
decreases from 0.85 to 0.27, from 0.38 to 0.25, and from
0.24 to 0.15 when CV was combined with AT using 5, 10,
and 25 ppm nanoparticles, respectively (Figs. 1B and 2B).
However, the synergistic effect of combining CV with AT
and/or PR was less than the effect of combining AT and
PR; the latter combination showed the strongest antifungal
activity. This result suggests that CV may interfere to some
extent with microbial absorption.

Effect of Silver Nanoparticles on Hyphae of 

Raffaelea

sp.

 

As described above, silver nanoparticles inhibited the growth
of fungal hyphae. In order to visualize the microscopic
effect of this treatment, healthy fungal hyphae grown on
MA plates were sprayed with 10 ppm AT solution, and
then observed under an electron microscope. Breakage of
hyphal tips, where new conidia form, as well as detached
conidia, were detected simultaneously (Fig. 3). Damage to
the surface of the fungal hyphae was also observed, which
could have caused the release of internal cellular materials,
resulting in shrinkage of the hyphae. Contrary to this
observation, hyphae treated with water appear to have
remained intact (Fig. 3).

Inhibition of Conidial Germination

The effect of silver nanoparticles on conidial germination
in 

Raffaelea

 sp. was assayed on Petri plates. Microscopic

observation revealed that conidial germination was inhibited
on plates containing silver nanoparticles, whereas actively
growing hyphae resulting from conidial germination were
clearly observed on plates treated with water only (Fig. 4).
Germination did not occur following prolonged incubation
of conidia treated with nanosilver (data not shown). 

Fig. 2.

 Effect of combining different forms of silver

nanoparticles on hyphal growth in 

Raffaelea 

sp. 

A

. Radial hyphal growth on MA medium containing indicated concentrations

of silver nanoparticle combinations. Pictures shown were taken at 7 days
post-inoculation. Three independent experiments were performed. 

B

.

 

Relative

hyphal growth rate on MA medium containing indicated concentrations of
silver nanoparticle combinations. Colony diameters were measured at
7 days post-inoculation. Data were obtained from triplicate assays; data are
presented as means

±

SD.

background image

763

Kim 

et al.

D

ISCUSSION

Recently, several microorganisms have threatened to cause
an ecological disaster due to the destruction of a variety of
tree species in many countries. Among them, ascomycetous
fungi belonging to the genus 

Raffaelea

 have become major

pathogens of trees, causing severe wilt disease. Along with
the outbreak of oak death caused by an unidentified 

Raffaelea

species in Korea, 

R. lauricola 

has caused the death of

nearly all redbay and sassafras trees in the U.S.A. since

2002 [5]. A symbiotic relationship between ambrosia
pathogens and their vectors is responsible for establishing
disease in diverse hosts. The outbreak of disease in redbay
trees is associated with a newly introduced ambrosia
beetle, 

Xyleborus glabratus 

[5]. In Korea, the oak wilt

pathogen 

Raffaelea

 sp. is mainly vectored by the beetle 

P.

koryoensis,

 whereas in Japan, 

R. quercivora

 is transferred

by 

P. quercivorus

 to oak species that are different from

those in Korea [11]. The rapid, potentially catastrophic
devastation that has occurred is considered to have resulted
from both the loss of biodiversity among tree species and
the introduction of foreign pathogens [5]. This dual threat
increases the likelihood that almost all oak trees could be
destroyed by pathogenic events. The wilt disease caused
by 

Raffaelea 

involves the dysfunction of sapwood [12]. In

response to a vector-mediated attack by the pathogen, the
host produces secondary metabolites that subsequently
cause cavitation or discoloration in the water-conducting
system. However, the host response is insufficient to kill
the pathogen or prevent its spread. Continued progression
of the fungal infection augments xylem dysfunction,
causing the death of the host. Unfortunately, there is no
known cure or means of controlling the disease.

It has been suggested that nanometer-sized silver particles

possess different physical and chemical properties from
their macroscale counterparts that alter their interaction
with biological structures and physiological processes [14].
Indeed, several pieces of evidence support the hypothesis
that silver nanoparticles have enhanced antimicrobial
activity. Silver nanoparticles are highly reactive because
they generate Ag

+

 ions, whereas metallic silver is relatively

unreactive [13]. It has also been shown that nanoparticles
efficiently penetrate microbial cells, suggesting that lower
concentrations of nanosized silver particles would be
sufficient for microbial control. This approach could be
more efficient than existing treatments, especially for
certain organisms that are less sensitive to antibiotics because
of their resistance to cell penetration [15]. In a previous
study, it was observed that silver nanoparticles disrupt
transport systems, including ion efflux [13]. Dysfunction
in ion efflux can cause rapid accumulation of silver ions,
interrupting cellular processes such as metabolism and
respiration by reacting with certain molecules. Moreover,
silver ions are known to produce reactive oxygen species
(ROS) that are detrimental to cells, causing damage to
proteins, lipids, and nucleic acids [7, 16]. 

Little is known regarding the effects of silver on

phytopathogenic fungi, because most studies have focused
on bacterial and viral pathogens in animals. Here, we
evaluated the antifungal activity of silver nanoparticles
against the fungal phytopathogen 

Raffaelea 

sp

.

 Our data

clearly demonstrated that the nanoparticles strongly
inhibited fungal growth and development and damaged
cell walls. These results suggest the possibility of using

Fig. 3.

 Electron micrographs of hyphae of 

Raffaelea

 sp. 

Fungal hyphae grown on MA plates were treated with either 10 ppm silver
nanoparticle solution AT or an equal volume of water as a control (Mock).
Representative photos were taken under SEM 72 h after treatment.

Fig. 4.

 Effect of silver nanoparticle solution AT on the

germination of conidia in 

Raffaelea 

sp. 

Conidia suspension (10

4

/ml) was spread on MA medium supplemented

with either water (Mock) or 10 ppm AT. Photos were taken under a light
microscope 48 h after conidium inoculation.

background image

E

FFECT

 

OF

 S

ILVER

 N

ANOPARTICLES

 

ON

 

A

 F

UNGAL

 P

HYTOPATHOGEN

R

AFFAELEA

 

SP

.

764

silver nanoparticles to eradicate phytopathogens. Several
parameters will require evaluation prior to practical application,
including phytotoxicity and antimicrobial effects 

in situ

,

and development of systems for delivering particles into
host tissues that have been colonized by phytopathogens. 

Acknowledgments

We thank Dr. Jong Kyu Lee at Kangwon National
University for providing the fungal species. This research
was supported by grants from the Korea Forest Service,
the Ministry for Food, Agriculture, Forestry and Fisheries,
and, in part, the Agriculture and Life Sciences Research
Institute (ALSRI) of Kangwon National University. 

R

EFERENCES

1. Batra, L. R. 1963. Ecology of ambrosia fungi and their

dissemination by beetles. 

Trans. Kansas Acad. Sci.

 

66:

 213

-

236.

2. Bragg, P. D. and D. J. Rannie. 1974. The effect of silver ions on

the respiratory chain of 

Escherichia coli

Can. J. Microbiol.

 

20:

883

-

889.

3. Elchiguerra, J. L., J. L. Burt, J. R. Morones, A. Camacho-

Bragado, X. Gao, H. H. Lara, and M. J. Yacaman. 2005.
Interaction of silver nanoparticles with HIV-1. 

J. Nanobiotechnol.

3:

 6.

4. Feng, Q. L., J. Wu, G. O. Chen, F. Z. Cui, T. N. Kim, and J. O.

Kim. 2000. A mechanistic study of the antibacterial effect of
silver ions on 

Escherichia coli 

and 

Staphylococcus aureus

J.

Biomed. Mater. Res

52:

 662

-

668.

5. Fraedrich, S. W. 2008. California laurel is susceptible to laurel

wilt caused by 

Raffaelea lauricola

Plant Disease

 

92:

 1469.

6. Gebhardt, H. and F. Oberwinkler. 2005. Conidial development

in selected ambrosial species of the genus 

Raffaelea

Antonie

van Leewenhoek

 

88:

 61

-

66.

7. Hwang, E. T., J. H. Lee, Y. J. Chae, Y. S. Kim, B. C. Kim, B. I.

Sang, and M. B. Gu. 2008. Analysis of the toxic mode of action
of silver nanoparticles using stress-specific bioluminescent
bacteria. 

Small

 

4:

 746

-

750.

8. Ito, S., T. Kubono, N. Sahashi, and T. Yamada. 1998.

Associated fungi with the mass mortality of oak trees. 

J. Japan

For. Soc.

 

80:

 170

-

175.

9. Jones, G. J. and M. Blackwell. 1998. Phylogenetic analysis of

ambrosial species in the genus 

Raffaelea

 based on 18S rDNA

sequences. 

Mycol. Res.

 

102:

 661

-

665.

10. Kinuura, H. 2002. Relative dominance of the model fungus,

Raffaelea

 sp., in the mycangium and proventriculus in relation

to adult stages of the oak platypodid beetle, 

Platypus

quercivorus

 (Coleoptera; Platypodidae). 

J. For. Res.

 

7:

 7

-

12.

11. Kinuura, H. and M. Kobayashi. 2006. Death of 

Quercus

crispula

 by inoculation with adult 

Platypus quercivorus

(Coleoptera: Platypodidae). 

Appl. Entomol. Zool

41:

 123

-

128.

12. Kuroda, K. 2001. Response of 

Quercus

 sapwood to infection

with the pathogenic fungus of a new wilt disease vectored by
the ambrosia beetle 

Platypus quercivorus

J. Wood Sci.

 

47:

425

-

429.

13. Morones, J. R., J. L. Elechiguerra, A. Camacho, K. Holt,

J. B. Kouri, J. T. Ramirez, and M. J. Yacaman. 2005. The
bactericidal effect of silver nanoparticles. 

Nanobiotechnology.

16:

 2346

-

2353.

14. Nel, A., T. Xia, L. Mädler, and N. Li. 2003. Toxic potential of

materials at the nanolevel. 

Science

 

311:

 622

-

627.

15. Samuel, U. and J. P. Guggenbichler. 2004. Prevention of

catheter-related infections: The potential of a new nano-silver
impregnated catheter. 

Int. J. Antimicrob. Agents

 

23S1:

 S75

-

S78.

16. Storz, G. and J. A. Imlay. 1999. Oxidative stress. 

Curr. Opin.

Microbiol

2:

 188

-

194.

17. Thurman, K. G. and C. H. P. Gerba. 1989. The molecular

mechanisms of copper and silver ion disinfection of bacteria
and viruses. 

Crit. Rev. Environ. Control

 

18:

 295

-

315.

18. Yeo, S. Y., H. J. Lee, and S. H. Jeong. 2003. Preparation of

nanocomposite fibers for permanent antibacterial effect. 

J.

Mater. Sci.

 

38:

 2143

-

2147.