J Gen Plant Pathol (2006) 72:348–350
DOI 10.1007/s10327-006-0300-1
TECHNIQUE
Ken-ichiro Saitoh · Kana Togashi · Tsutomu Arie
Tohru Teraoka
A simple method for a mini-preparation of fungal DNA
Received: January 27, 2006 / Accepted: March 31, 2006
Abstract A simple method was established to prepare
DNA from fungal mycelia cultured on an agar plate. The
fungi tested successfully with this method contained Zygo-
mycetes, Ascomycetes, Basidiomycetes, and Oomycetes.
This method did not require any time-consuming steps to
crush or digest mycelia or fractionation in a phenol–chloro-
form mixture. The DNA was easily extracted by immersing
and dispersing the mycelial plugs in a specific buffer
(200 mM Tris-HCl, 50 mM ethylenediaminetetraacetic acid,
200 mM NaCl, 1% n-lauroylsarcosine, pH 8.0), then concen-
trated by ethanol precipitation. The total time to complete
the whole procedure was less than 1 h. The quality and
quantity were sufficient for polymerase chain reaction
amplification and Southern blot analysis.
Key words Fungal DNA extraction · Zygomycetes ·
Ascomycetes · Basidiomycetes · Oomycetes
Extraction of genomic DNA is an essential step for molecu-
lar analyses of fungi. The standard method to prepare fun-
gal DNA consists of lyophilization of mycelia, disruption of
cell walls by grinding, extraction of DNA in buffers contain-
ing sodium dodecyl sulfate, removal of proteins with a mix-
ture of phenol and chloroform, and precipitation of DNA
with 2-propanol (Kawabe et al. 2004). This method is suit-
able to obtain a large amount of pure DNA, but is time
consuming, labor intensive, and pollutes water with phenol
and chloroform. Therefore, a rapid and simple method for
DNA preparation on a small scale has been needed for
polymerase chain reaction (PCR)-based identification and
K. Saitoh · K. Togashi · T. Arie · T. Teraoka
Graduate School of Agriculture, Tokyo University of Agriculture
and Technology, Tokyo, Japan
T. Teraoka (*)
Laboratory of Plant Pathology, Faculty of Agriculture, Tokyo
University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu,
Tokyo 183-8509, Japan
Tel./Fax +81-42-367-5692
e-mail: teraoka@cc.tuat.ac.jp
© The Phytopathological Society of Japan and Springer 2006
determination of genotypes such as mating types or drug
resistance, and for screening transformants to obtain iso-
lates with a targeted gene modification. Generally, a fungus
isolated from the field or a mixture of transformants is
grown in liquid culture before DNA extraction. If DNA can
be successfully extracted directly from thalli on agar media,
the total time for the identification or screening will be
markedly decreased. Several methods for rapid DNA ex-
traction have been proposed (Cenis 1992; Ferreira and
Glass 1996; Griffin et al. 2002; Xu and Hamer 1995), but
they were not fully suitable for this purpose.
In this report, a simple and reliable method was devel-
oped for the mini preparation of fungal DNA from thalli on
agar media, based on the method of Liu et al. (2000). This
method has several advantages:
1. Preculture in liquid media is not necessary.
2. The duration of culture is more flexible and can be cho-
sen before the preparation.
3. Potentially harmful organic solvents, such as phenol and
chloroform, which are troublesome to discard, are ex-
cluded as exhaustively as possible.
4. DNA is less damaged by mechanical forces because
freezing and homogenizing the thalli are not needed.
5. The minimized number of preparation steps and the lyo-
philized powder-free procedure reduce the risk of cross-
contamination.
6. Total time of the procedure is less than 1 h.
Magnaporthe grisea was cultured on Misato-Hara agar
medium (0.2% yeast extract, 1% soluble starch, 1.5% agar)
at 26°C. Fusarium oxysporum was cultured on potato-
sucrose agar medium [20% (w/v) potato extract, 0.5% su-
crose, 1.5% agar] at 26°C. A small piece of mycelia (7–
15 mm on a side) with agar medium (100–300 mg total mass)
was excised with a toothpick or an inoculation needle from
a 4- to 10-day-old culture plate (Fig. 1A) and transferred to
a microtube with 500 µl of the lysis buffer (200 mM Tris-
HCl, 50 mM ethylenediaminetetraacetic acid, 200 mM
NaCl, 1% n-lauroylsarcosine sodium salt, pH 8.0). The
mycelia were dispersed in the buffer with the toothpick and
incubated for 10 min at room temperature. The mixture was
 349

then centrifuged at 18 000 g (15 000 rpm in a T15AP21 rotor,
Hitachi Koki, Tokyo, Japan) for 5 min at 4°C, and the super-
natant (300 µl) was transferred to a fresh microtube. After
mixing with 750 µl of ethanol by inverting the tube, the
DNA was precipitated by centrifugation at 18 000 g for
2 min at 4°C. After a wash with 70% ethanol, the DNA
pellet was air-dried and dissolved in 50 µl of TE buffer (pH
8.0).
To check the quality and quantity of the DNA prepared
by this method, 5 µl of the DNA solution was electrophore-
sed on 0.8% agarose gel and stained with ethidium bromide
(Fig. 1B). About 50–200 ng of DNA were obtained from M.
grisea isolate P2 and F. oxysporum f. sp. lycopersici isolate
880621a-1 by this method. In these DNA samples, RNAs
were probably degraded; they were hardly detected at more
than 500 bp (Fig. 1B).
DNA prepared by this method was used as a template
for PCR to amplify the internal transcribed spacer (ITS)
region. PCR primers were ITS1 (5′-TCCGTAGGTGAA
CCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGAT
ATGC-3′) (White et al. 1990). Isolates of fungal species
within the Zygomycota, Ascomycota, Basidiomycota, or
Oomycota (Chromista) used in this experiment are listed in
Table 1. All isolates were cultured on potato-sucrose agar
medium at 26°C, except for Phytophthora infestans grown
on rye agar medium [6.7% (w/v) rye extract, 2% sucrose,
0.35% agar] at 20°C. The PCR mixture (20 µl) contained
0.5 U Taq DNA polymerase (Sigma Aldrich, St. Louis, MO,
USA), 1 × PCR buffer (Sigma Aldrich), 2.5 mM MgCl2,
0.2 mM (each) dNTPs, 0.4 µM (each) primers, and 1 µl of the
prepared DNA. Thermal conditions were as follows: dena-
turing at 94°C for 2 min; 30 cycles of 94°C for 30 s, 57°C for
30 s, 72°C for 1 min; and a final extension at 72°C for 7 min.
Fragments of the ITS region were successfully amplified
from all tested isolates as expected (Fig. 2).
The quality of the DNA obtained by this method was
sufficient for digestion by restriction endonucleases, and the
extraction protocol can be easily scaled up. The DNA
preparations in this case were used in Southern blot analy-
ses (Fig. 3).

Table 1. Fungal species and isolates used for DNA preparation

Species Isolate Reference

A B (kbp) λ Mg Fo Zygomycetes
Mucor sp. 0016-2-2 Okabe et al. (2005)
23.1 - Ascomycetes and
9.4 - mitosporic Ascomycetes
Magnaporthe griesa P2 Saitoh et al. (2003)
4.4 - Aspergillus oryzae RIB40 Machida et al. (2005)
Penicillium sp. 0044-1-2 Okabe et al. (2005)
2.0 - Botryotinia fuckeliana 031024d Takahashi et al. (2005)
Sclerotinia sclerotiorum 910624a Takahashi et al. (2005)
Fusarium oxysporum 880621a-1 Kawabe et al. (2004)
f. sp. lycopersici
Gibberella fujikuroi FGSC 7611 Arie et al. (1999)
Trichoderma sp. 0039-1-6 Okabe et al. (2005)
Cladosporium sp. 0043-1-3 Okabe et al. (2005)
Alternaria sp. 0043-2-5 Okabe et al. (2005)
0.6 - Ulocladium sp. 0036-1-5 Okabe et al. (2005)
Basidiomycetes
Fig. 1A, B. Mini preparation method. A Excision of a mycelial block Athelia rolfsii 010829a-1 Takahashi et al. (2005)
on agar medium to start the mini preparation and B agarose gel (0.8%) Rhizoctonia solani TKF-44 Shigemoto et al. (1992)
electophoresis of the DNA prepared by this method from Oomycetes (Chromista)
Magnaporthe grisea (Mg) and Fusarium oxysporum f. sp. lycopersici Phytophthora infestans TKF-411 Takahashi et al. (2005)
(Fo). Lambda DNA digested (50 ng) with HindIII was used as a DNA Phytophthora nicotianae TPS1234 Umezawa et al. (2004)
molecular marker (l)

1 2 3 4 5 6 7 8 99 10 11 12 13 14 15 16 M

- 1000 bp

- 500 bp

- 100 bp

Fig. 2. Polymerase chain reaction (PCR) amplification using DNA
from mini preparation. The prepared DNA from Mucor sp. (lane 1),
Magnaporthe grisea (lane 2), Aspergillus oryzae (lane 3), Penicillium
sp. (lane 4), Botryotinia fuckeliana (lane 5), Sclerotinia sclerotiorum
(lane 6), Fusarium oxysporum f. sp. lycopersici (lane 7), Gibberella
fujikuroi (lane 8), Trichoderma sp. (lane 9), Cladosporium sp. (lane
10), Alternaria sp. (lane 11), Ulocladium sp. (lane 12), Athelia rolfsii
(lane 13), Rhizoctonia solani (lane 14), Phytophthora infestans (lane
15), and Phytophthora nicotianae (lane 16) were used as templates. The
PCR mixture and reaction conditions are described in the text. A
sample (1 µl) of the reaction mixture (20 µl) was separated by electro-
phoresis using a 1.5% agarose gel. Primers used for the internal tran-
scribed spacer region are described in the text. Lane M is a 100-bp
DNA ladder size marker (New England Biolabs, Ipswich, MA, USA)
350
A 1 kb
Mg-NCS-1
P E P tion. Nucleic Acids Res 20:2380
B Griffin DW, Kellogg CA, Peak KK, Shinn EA (2002) A rapid and
(kbp) P E
9.4
6.6
4.4
2.0
Fig. 3A, B. Southern blot analysis using the genomic DNA from the
mini preparation. A Mg-NCS-1 probe labeled with DIG was prepared
with a standard method using DIG DNA labeling and detection kit
(Roche Diagnostics, Manheim, Germany). A Genomic structure
around Mg-NCS-1 (Saitoh et al. 2003) in Magnapothe grisea isolate P2
is presented with recognition sites for PstI (P) and EcoRV (E). The
dotted line indicates the fragment used to prepare the probe. B The
genomic DNA (10 µl) prepared from M. grisea was digested with PstI
(P) or EcoRV (E), separated on 0.8% agarose gel, and transferred to
a nylon membrane (positively charged, Roche Diagnostics). DNA
fragments that hybridized with the probe were detected with 4-
nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate
(Roche Diagnostics). These procedures were carried out as described
in the manufacturer’s protocol
This method was successfully used with mycelia cultured
on diverse media such as oatmeal agar, V-8 agar, and po-
tato-dextrose agar. Although the quality of the DNA after
long-term storage has not yet been checked, the DNA was
stable enough for use as a template for PCR after storage
for 3 weeks at 4°C.
Acknowledgments We thank Mr. Ryo Ishikawa (Sumitomo Chemical
Co., Ltd., Hyogo, Japan), Ms. Junko Umezawa Sekihara (Toyama
Agricultural Research Center, Toyama, Japan), and Dr. Michio
Takeuchi (Tokyo University of Agriculture and Technology, Tokyo,
Japan) for providing the isolates. This study was supported in part by a
Grant-in-Aid for Scientific Research (A) from the Ministry of Educa-
tion, Science, Sports, and Culture, Japan (no. 16208004).
References
Arie T, Yoshida T, Shimizu T, Kawabe M, Yoneyama K, Yamaguchi I
(1999) Assessment of Gibberella fujikuroi mating type by PCR.
Mycoscience 40:311–314
Cenis JL (1992) Rapid extraction of fungal DNA for PCR amplifica-
Ferreira AVB, Glass NL (1996) PCR from fungal spores after micro-
wave treatment. Fungal Genet Newsl 43:25–26
efficient assay for extracting DNA from fungi. Lett Appl Microbiol
34:210–214
Kawabe M, Mizutani K, Yoshida T, Teraoka T, Yoneyama K,
Yamaguchi I, Arie T (2004) Cloning of the pathogenicity-related
gene FPD1 in Fusarium oxysporum f. sp. lycopersici. J Gen Plant
Pathol 70:16–20
Liu D, Coloe S, Baird R, Pedersen J (2000) Rapid mini-preparation of
fungal DNA for PCR. J Clin Microbiol 38:471
Machida M, Asai K, Sano M, Tanaka T, Kumagai T, Terai G,
Kusumoto K, Arima T, Akita O, Kashiwagi Y, Abe K, Gomi K,
Horiuchi H, Kitamoto K, Kobayashi T, Takeuchi M, Denning DW,
Galagan JE, Nierman WC, Yu J, Archer DB, Bennett JW,
Bhatnagar D, Cleveland TE, Fedorova ND, Gotoh O, Horikawa H,
Hosoyama A, Ichinomiya M, Igarashi R, Iwashita K, Juvvadi PR,
Kato M, Kato Y, Kin T, Kokubun A, Maeda H, Maeyama N,
Maruyama J, Nagasaki H, Nakajima T, Oda K, Okada K, Paulsen I,
Sakamoto K, Sawano T, Takahashi M, Takase K, Terabayashi Y,
Wortman JR, Yamada O, Yamagata Y, Anazawa H, Hata Y, Koide
Y, Komori T, Koyama Y, Minetoki T, Suharnan S, Tanaka A, Isono
K, Kuhara S, Ogasawara N, Kikuchi H (2005) Genome sequencing
and analysis of Aspergillus oryzae. Nature 438:1157–1161
Okabe A, Ishikawa N, Kawabe M, Kodama M, Teraoka T, Arie T
(2005) Genealogical analysis of Fusarium oxysporum isolates from
wild Lycopersicon spp. in Chili (in Japanese). Jpn J Phytopathol
71:218–219
Saitoh K, Arie T, Teraoka T, Yamaguchi I, Kamakura T (2003) Tar-
geted gene disruption of the neuronal calcium sensor 1 homologue in
rice blast fungus, Magnaporthe grisea. Biosci Biotechnol Biochem
67:651–653
Shigemoto R, Okuno T, Matsuura K (1992) Effects of validamycin A
on the growth of and trehalose content in mycelia of Rhizoctonia
solani incubated in a medium containing several sugars as the sole
carbon source. Ann Phytopathol Soc Jpn 58:685–690
Takahashi H, Shimizu A, Arie T, Rosmalawati S, Fukushima S,
Kikuchi M, Hikichi Y, Kanda A, Takahashi A, Kiba A, Ohnishi K,
Ichinose Y, Taguchi F, Yasuda C, Kodama M, Egusa M, Masuta C,
Sawada H, Shibata D, Hori K, Watanabe Y (2005) Catalog of Micro-
Tom tomato response to common fungal, bacterial, and viral patho-
gens. J Gen Plant Pathol 71:8–22
Umezawa J, Morikawa T, Mukobata T, Saitoh K (2004) Difference of
cultivar resistance and effective chemicals to Phytophthora basal rot
of welsh onion caused by Phytophthora nicotianae (in Japanese). Jpn
J Phythopathol 70:212
White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics. In:
Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols:
a guide to methods and applications. Academic, New York, pp 315–
322
Xu JR, Hamer JE (1995) Assessment of Magnaporthe grisea mating
type by spore PCR. Fungal Genet Newsl 42:80