Microb. Resour. Syst. 37
June
（1）
：1
2021
─ 12, 2021

Vol. 37, No. 1

Lecanicillium aphanocladii isolated from Tengu-no-Mugimeshi
found in Mount Kurohime, Nagano Prefecture, central Japan
Mayuka Higo1), Hiroki Kojima1), Masato Iwatsuki1,2), Tomotaka Tanabe3),
Takashi Yaguchi4), Toru Okuda5), Taiga Kasuya6) and Kenichi Nonaka1,2)＊
Ōmura Satoshi Memorial Institute, 2)Graduate School of Infection Control Sciences, Kitasato University
5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan3)
Togakushi Museum of Natural History, 3400 Togakushi Tochihara, Nagano 381-4104, Japan
4)
Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan
5)
HyphaGenesis Inc., 2-18-28 Tamagawa-Gakuen, Machida, Tokyo 194-0041, Japan
6)
Department of Biology, Keio University, 4-1-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8521, Japan1)

Tengu-no-Mugimeshi is a soil-like mass of microorganisms that was collected on Japan’s Mount Kurohime in 1939. It
was sealed in glass bottles and preserved at an elementary school in Nagano City for 80 years. Upon opening the bottle,
a single verticillium-like fungal strain, FKI-9593 (=IFM 64743), was isolated from the preserved material. Using a
combination of micro-morphological characteristics and multigene phylogeny (SSU, LSU and TEF), the isolate was
identified as Lecanicillium aphanocladii. A culture broth of the organism was used to test for antimicrobial activity
against 13 selected microbes. As a result, weak antibacterial activity against Proteus vulgaris NBRC 3167 was confirmed.
Structural analysis identified the active compound as the polyketide metabolite, oosporein.

Key words: Ascomycota, multi-gene phylogeny, antibacterial activity, secondary metabolite

INTRODUCTION

There have been some previous reports on the constituent microbes of Tengu-no-Mugimeshi (Kawamura,
1916; Ono, 1916; Yagi, 1936).
In 1902, Bacillus sp., Verticillium sp., Leucosystis
sp. and a species of“Kapselbacterien”were isolated
from Tengu-no-Mugimeshi collected on Mount
Kurohime in the northern part of Nagano Prefecture(Ono, 1916). In 1914, species of the ‘Oomycetes’,Bacillus fluorescens and Penicillium sp. were isolated from Tengu-no-Mugimeshi collected on the same
mountain (Kawamura, 1916). In 1926, all bacteria isolated from Tengu-no-Mugimeshi were cyanobacteria(Molisch, 1926). Although there were multiple reportsof bacteria isolated from Tengu-no-Mugimeshi, few
research reports described fungi as components ofthe mixture.
One site named as“Miso-zuka”in Mikageshinden,
Komoro of Nagano Prefecture where Tengu-noMugimeshi is found is protected as a nationally protected natural monument (Yagi, 1936). Among theareas where Tengu-no-Mugimeshi is commonly
found, known localities of Tengu-no-Mugimeshi at
Mt. Kurohime and Mt. Iizuna are strictly protected
as Special Protection Zones in Myoko-Togakushi
Renzan National Park by Japan’s Ministry of the

Tengu-no-Mugimeshi is an aggregation of microbial material usually consisting of up to 10 types of
microbes; Eubacteria, Archaea, Cyanobacteria,
Ktedonobacteria and Fungi (Wang et al., 2019).
“Tengu”is a kind of demigod popular in Japanesefolklore;“Tengu-no-mugimeshi”means boiled barleyof Tengu (Okada, 1937). It is orange-to-brown incolor, granular and very similar to soil in appearance,
and is also referred to as“eatable soil”(Okada, 1937).
Geologically speaking, Tengu-no-Mugimeshi is foundin a graben, called the Fossa Magna, a great rift lowland that traverses the widest section of Honshu
from the Sea of Japan to the Pacific (Motojima et al.,1955). It is only found in volcanic areas above 2000
m in altitude on the Fossa Magna. Geographically,
this area is located in central Japan and lies in the
Chubu and northern Kanto regions (Motojima et al.,1955). It is known that the constituent microbes ofTengu-no-Mugimeshi vary depending on the region.
Corresponding author＊

E-mail: ken@lisci.kitasato-u.ac.jp
Accepted: November 28, 2020
─1─The constituent fungi of Tengu-no-Mugimeshi stored for 80 years

Higo et al.

Environment, so samples cannot be collected without special permits. The specimen of Tengu-noMugimeshi examined in this study was collectedfrom the summit of Mt. Kurohime in 1939. This
specimen was preserved in a sealed glass bottle andmaintained at Terao Elementary School, Nagano
City, Japan for over 80 years (Fig. 1). This bottle
was subsequently rediscovered in 2016 and is now
kept at this Togakushi Museum of Natural History,
Nagano City, Japan. Historically, few fungi havebeen isolated from Tengu-no-Mugimeshi, therefore,
we report herein details of a fungal strain isolated
from this 1939 sample, including notes on its taxonomy, phylogeny and secondary metabolite profile.

MATERIALS AND METHODS

Isolation
To isolate fungi, a sample of Tengu-no-Mugimeshi(1 g) taken from the aforementioned specimen was
suspended in 9 ml of modified Winogradsky’s salt
s o l u t i o n (0.38% K 2 H P O 4 , 0.12% K H 2 P O 4 , 0.51%
MgSO4･7H2O, 0.25% NaCl, 0.005% Fe2 (SO4)3･nH2O
and 0.005% MnSO 4･5H 2O) with 0.01% Tween-80(Sigma-Aldrich Co., Saint Louis, MO, USA) and thensonicated for 2 min, and diluted to 102－103 fold withthe above Winogradsky’s salt solution. Aliquots ofdiluted suspension totaling 200 ml were spread on
Petri dishes with solidified potato dextrose agar (PDA,
Difco Laboratories Ltd., Detroit, MI, USA), malt
extract agar (MEA; Pitt, 1979) and Czapek yeast
extract agar (CYA; Pitt, 1979), and all media with50 mg/l rose bengal, 100 mg/l kanamycin and chloramphenicol. To isolate bacteria from Tengu-noMugimeshi, PDA without antibiotics was used.
Plates were incubated at 25°C for 7 days. A fungus
was then isolated and the isolate was preserved as
FKI-9593 at the Ōmura Satoshi Memorial Institute,
Kitasato University, and as IFM 64743 at the
Medical Mycology Reseach Center, Chiba University
through the National Bio-Resource Project.

A glass bottle containing Tengu-no-Mugimeshi preserved at Terao Elementary School, Nagano City,Japan

Tengu-no-Mugimeshi showing the granular mass
appearance
Fig. ₁Morphological analysis
To observe morphological characteristics, the isolate was incubated on PDA, MEA and potato carrot
agar (PCA; Atlas, 2010) at 25°C (also at 5°C and 37°C
on PDA) for 7 days in the dark. Colony colours
were designated according to Kornerup & Wanscher(1978).
Determination of micro-morphological characteristics followed as reported by Zare & Gams (2001) andStudied sample of Tengu-no-Mugimeshi

Kaifuchi et al. (2013). For observation of micro-morphological characteristics, microscope slides wereprepared from PCA (Atlas, 2010) cultures. The
slides were examined with a Vanox-S AH-2 microscope (Olympus Co., Ltd., Tokyo, Japan), and digital
photomicrographs were taken with a DP25 digital
camera (Olympus Co., Ltd.).
For scanning electron microscopy (SEM), agar
─2─Microb. Resour. Syst. June 2021

Vol. 37, No. 1

blocks (5×5 mm) cultured on PCA and MEA medium for 7 days were prepared. The samples were
air-dried. Sputter-coating with gold was accomplished using a JFC-1200 Fine Coater (JEOL Ltd.,Tokyo, Japan). These samples were observed with a
JSM-5600 scanning electron microscope (JEOL Ltd.).

dant with each other, we describe the phylogenetic
results based on the ML tree (Fig. 2). There was no
topological conflict among the SSU tree, LSU tree
and TEF tree for highly-supported clades, and theconcatenated tree was more robust than the separated ones. The alignments and trees were deposited in TreeBASE (http://www.treebase.org/) withthe accession number 26425.

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted using the PrepMan
Ultra® Sample Preparation Reagent (Thermo Fisher
Scientific, Inc., Waltham, MA, USA), according to the
manufacturer’s instructions. Portions of DNA of
nuclear ribosomal small and large subunit (SSU and
LSU) DNA and elongation factor 1α (TEF) gene,
were amplified by polymerase chain reaction (PCR)
conducted under the conditions detailed by Nonaka
et al.(2013). Sequencing reaction was performed
using a BigDye® Terminator according to the manufacture’s instructions, then sequence products were
purified using a BigDye® XTerminator Purification
Kit (Thermo Fisher Scientific, Inc.). And samples
were analyzed on an ABI PRISM 3130 Genetic
Analyzer (Thermo Fisher Scientific, Inc.). Contigs
were assembled using the forward and reverse
sequences, with the SeqMan Pro program from theLasergene 10 package (DNASTAR Inc., Madison,
WI, USA). The sequences were deposited at the
GenBank (Table 1).

Fermentation of fungal strain
To assay for antimicrobial activity of culture
broths against representative microbes, as well toenable metabolite analyses, the strain was inoculated
into 10 ml of seed medium containing: 2.0% glucose,
0.5% Hipolypepton (Nihon Pharmaceutical Co., Ltd.,Tokyo, Japan), 0.2% yeast extract, 0.2% KH2PO4,
0.05% MgSO4･7H2O, 0.1% agar; adjusted to pH 6.0
before sterilization. The test tube containing the
mixture was incubated on a rotary shaker (300 rpm)
at 27°C for three days. A 1-ml portion of the seed
culture of FKI-9593 was transferred to three 500-ml
Erlenmeyer flasks containing 100 ml of F38 production medium (3.0% sucrose, 3.0% soluble starch, 1.0%
malt extract, 0.3%“Ebios”, a commercial product ofdried yeast by Asahi Group Foods, Ltd. (Tokyo,
Japan), 0.5% KH2PO4, 0.05% MgSO4･7H2O; adjusted
to pH 6.0 before sterilization), and the fermentation
was carried out on a rotary shaker (210 rpm) at 27°C
for three days and then stationary for 10 days. The
culture broth (300 ml) was mixed with ethanol(300 ml), followed by centrifugation (3,000 rpm for 10
min).

Phylogenetic analysis
Sequences of all regions were individually aligned
using MUSCLE 3.8.31 (Edgar, 2004). The alignments
were refined using SeaView 4.7 (Gouy et al., 2010).
The total length of aligned sequences was 2,476
nucleotides. Three-genes (SSU, LSU and TEF) wereconnected into a single dataset. Before combining
the data sets, each set was analysed using the neighbor-joining (NJ) method (Saitou & Nei, 1987).
Phylogenetic analyses of the 3-genes dataset werebased on the maximum-parsimony (MP) method
using PHYLIP’s dnapars algorithm, the NJ method
and the maximum likelihood (ML) method using
PhyML 3.0 (Guindon et al., 2010) with GTR substitution model as implemented in SeaView 4.7 program.
Bootstrap analyses were performed on MP, NJ and
ML trees with 1,000 bootstrap replicates. The trees
were rooted with species of Simplicillium Zare & W.
Gams species according to Su et al.(2019) and Zhou
et al.(2018), and viewed in SeaView 4.7 program.
Because MP, NJ and ML trees were almost concor-

Instrumental analysis of secondary metabolites
The mixtures were evaluated by analytical HPLC
under the following conditions for the analysis of
metabolites: column, Symmetry C18 (2.1 ø×150 mm,
Waters Co., Tokyo, Japan); mobile phase, acetonitrilewater with 0.05% phosphoric acid, 5-100% (20 min);flow rate, 0.2 ml min－1; detection, UV at 210 nm.
Antimicrobial activities of culture broths against
representative microbes
Antimicrobial activity was measured using thepaper disc method (6 mm and 8 mm, Toyo Roshi
Kaisha Ltd., Tokyo, Japan). Each sample contained
10 μl and 50 μl of 50% ethanol extract of pure culture
broth of strain FKI-9593. Culture conditions were as
follows: Bacillus subtilis ATCC 6633 (Nutrient agar
(0.5% peptone, 0.5% meat extract and 0.8% agar),
─3─

─4─

CBS 350.85
CBS 797.84
FKI-9593＊
CBS 345.37
CBS 342.80
CBS 357.80

L. antillanum
L. aphanocladii

Cuba
China
Nagano, Japan
Germany
Villeurbanne, France
Netherlands

－
－
－
－
－
Amazon－－－Ex-typeTaxonomical
information

Coccus viridis－
Trialeurodes vaporariorum
Epitype－－
Parthenolecanium sp.
Holotype－－
Subfamily archolophinay－(Tineidae, Lepidoptera)
Soil under Damnacanthus indi︲ －
cus
－
－
－
－
Spider (Arachnida)－
Spider (Arachnida)－－－
Crinipellis perniciosa inEx-type
Theobroma cacao
AgaricEx-type
Agaricus bisporus－
Tengu-no-Mugimeshi－
Puccinia coronata－
Abies alba
Ex-type
Agaricus bisporus
Ex-type－
Leaf litter of Acer saccharum
Spider (Arachnida)
Icerya purchasi

Isolation source
SSU

AF339585
KM283763
LC553289
KM283764
KM283766
KM283767

MF416588
MF416602
EU369099
EU369090
MF416583
KM283762

LC553288

KM283770
KM283774
MF416573
KC633251
MF416600
AY184974

MF416572
AF339614
EU369109
KM283773

AF339536
KM283787
LC553284
KM283788
KM283790
KM283791

MF416534
MF416548
EU369035
EU369031
MF416530
KM283786

LC553283

KM283794
KM283798
MF416521
KC875225
MF416546
AY184963

MF416520
AF339565
EU369043
KM283797

LSU

TEF

DQ522350
KM283811
LC553294
KM283812
KM283814
KM283815

MF416478
MF416492
EU369018
EU369008
MF416473
KM283810

LC553293

DQ522359
KM283821
MF416466
KC633266
MF416490
EF469059

MF416465
EF468782
EU369026
KM283820

GenBank accession no.

＊

New isolates sequenced in this study. ANDES-F, Culture collection of the Museo de Historia Natural of the Universidad de los Andes in Bogotá, Colombia; BCC,
BIOTEC Culture Collection Laboratory, Klong Luang, Thailand; CBS, CBS-KNAW Culture Collection, Westerdijk Fungal Biodiversity Institute, Utrecht, The
Netherlands.; FKI, Fungal Collection of Ōmura Satoshi Memorial Institute, Kitasato University, Tokyo, Japan; KACC, Korean Agricultural Culture Collection,
National Institute of Agricultural Biotechnology, Rural Development Administration, Suwon, Republic of Korea; KUMCC, Culture collection of Kunming Institute ofBotany, Kunming, China; NHJ, Nigel Hywel-Jones personal collection; OSC, Oregon State University Herbarium, Corvallis, Oregon, USA

L. dimorphum
L. flavidum
L. fungicola
var. aleophilum

BCC 1919
BCC 16025
NHJ 10808
NHJ 10469
BCC 2093
CBS 418.81

FKI-7101

Blackwellomyces sp.

B. pseudomilitaris
Gibellula leiopus
G. pulchra
Hevansia arachnophilus
H. nelumboides
Lecanicillium acerosumWest Indies
UK－
Colombia－
Tennessee, USA

CBS 101247
CBS 143.62
HUA 186131
ANDES-F 1024
BCC 16819
OSC 93610
Kozu Islands, Japan－USA－
Sri Lanka

Locality

HUA 186145
CBS 402.78
NHJ 5112
CBS 126.27

Strain no.

ʟｅｃａｎｉｃｉˡˡｉｕⅿ isolate and related taxa used for phylogenetic analyses

Akanthomyces aculeatus
A. attenuatum
A. coccidioperitheciata
A. dipterigenus(Lecanicillium longisporum)
A. lecanii
A. muscarium
A. pistillariaeformis
A. sabanense
A. tuberculatus
Blackwellomyces cardinalis

Species

Table ₁

The constituent fungi of Tengu-no-Mugimeshi stored for 80 years
Higo et al.

─5─

CBS 101237
CBS 704.86
CBS 311.74
CBS 116.25
CBS 726.73a

L. primulinum
L. psalliotae
L. restrictum
L. subprimulinum
L. tenuipes
L. testudineum
L. uredinophilum

L. wallacei

Simplicillium lanosoniveum
S. obclavatum
S. lamellicola
Torubiella alba

Hemileia vastatrix
Air above sugarcane field
Agaricus bisporus
Araneida

Coltricia perennis
Soil under Alpinia zerumbet
Soil under Tarenna subsessilis
Soil under Carex oahuensis
Soil under unidentified plant
Soil
Wooden handle of a tool
Dead wood
SpiderCarapace of the turtle
Pucciniastrum agrimoniae on
Agrimonia pilosa
Lepidopteran larva

Agaricus bisporus

Isolation source

Continued－
Ex-typeEx-type
EpitypeHolotype

Ex-type－－－Ex-type－Ex-type－－Ex-type－

EpitypeTaxonomical
information

AF339602
AF339567
AF339601
AF339586

AY184978EF469128
LT548279
MG585316
KM283778
LT548278
KM283758

KM283769
LC553285
LC553286
LC553287

AF339553
AF339517
AF339552
AF339537

AY184967

AF339549
LC553280
LC553281
LC553282
AB712263
AF339558
LT548279
MG585315
KM283802
LT548278
KM283782

KM283792

LSU

TEF

DQ522358
EF468798
DQ522356
EF468781

EF469073

EF469066
LT626943
MG585317
DQ522341
LT626942
KM283806

KM283817
LC553290
LC553291
LC553292

KM283816

GenBank accession no.
KM283768

SSU＊

New isolates sequenced in this study. ANDES-F, Culture collection of the Museo de Historia Natural of the Universidad de los Andes in Bogotá, Colombia; BCC,
BIOTEC Culture Collection Laboratory, Klong Luang, Thailand; CBS, CBS-KNAW Culture Collection, Westerdijk Fungal Biodiversity Institute, Utrecht, The
Netherlands.; FKI, Fungal Collection of Ōmura Satoshi Memorial Institute, Kitasato University, Tokyo, Japan; KACC, Korean Agricultural Culture Collection,
National Institute of Agricultural Biotechnology, Rural Development Administration, Suwon, Republic of Korea; KUMCC, Culture collection of Kunming Institute ofBotany, Kunming, China; NHJ, Nigel Hywel-Jones personal collection; OSC, Oregon State University Herbarium, Corvallis, Oregon, USA

Dumoga Bone forest,
Sulawesi, Indonesia
Venezuela
India
UK
Ghana

Netherlands
Hatoma-jima, Japan
Haha-jima, Japan
Haha-jima, Japan
Okinawa, Japan
UK
Czech Republic
Yunnan, China
Spain
Czech Republic－

CBS 164.70
FKI-6310
FKI-6826
FKI-6887
FKI-6172
CBS 101270
CBS 143072
KUMCC17-0148
CBS 309.85
CBS 141096
KACC 44082

Locality
Netherlands

Strain no.
CBS 992.69

L. fungicola
var. fungicola
L. fusisporum
Lecanicillium sp.

Species

Table ₁

Microb. Resour. Syst. June 2021
Vol. 37, No. 1

The constituent fungi of Tengu-no-Mugimeshi stored for 80 years

Higo et al.

Fig. ₂ A phylogenetic tree for ʟｅｃａｎｉｃｉˡˡｉｕⅿ ａｐʰａｎｏｃˡａｄｉｉ and related species of the genus ʟｅｃａｎｉｃｉˡˡｉｕⅿ, drawn
from maximum likelihood analysis of multigene (SSU, LSU, TEF) sequences. The outgroup is three
Simplicillium (Cordycipitaceae) species. The numbers shown in the branches represent bootstrap values
exceeding 50% (MP, left/NJ, center/ML, right) based on 1,000 replicates. The strain numbers with a superscript
letter T mean ex-type strains.

─6─

Microb. Resour. Syst. June 2021

Vol. 37, No. 1

1.0% inoculation, 37°C for 24 h), Staphylococcus aureus
ATCC 6538p (Nutrient agar, 0.2% inoculation, 37°C
for 24 h), Kocuria rizophila ATCC 9341 (Nutrient
agar, 0.2% inoculation, 37°C for 24 h), Mycobacterium
smegmatis ATCC 607 (Waksman agar (0.5% peptone,
0.5% meat extract, 0.3% NaCl, 1.0% glucose and 0.8%agar), 1.0% inoculation, 37°C for 24 h), Escherichia
coli NIHJ (Nutrient agar, 0.5% inoculation, 37°C for24 h), Pseudomonas aeruginosa IFO 3080 (Nutrient
agar, 1.9% inoculation, 37°C for 24 h), Xanthomonas
oryzae KB 88 (Nutrient agar, 1.0% inoculation, 27°C
for 24 h), Proteus vulgaris NBRC 3167 (Nutrient agar,
0.5% inoculation, 37°C for 24 h), Klebsiella pneumoni︲ae ATCC 10031 (Nutrient agar, 0.5% inoculation, 37°C for 24 h), Candida albicans ATCC 64548 (GY agar,
0.2% inoculation, 27°C for 24 h), Saccharomyces cere︲visiae ATCC 9763 (GY agar, 0.3% inoculation, 27°C
for 24 h), Aspergillus niger ATCC 6275 (GY agar,
0.3% inoculation, 27°C for 48 h), Mucor racemosus
IFO 4581 (GY agar, 0.3% inoculation, 27°C for 48 h).

diameter after 7 days at 25°C, very raised, covered
with white (1A1), reverse dull red (8C3). Colonies on
PCA 50 mm in diameter after 7 days at 25°C, white,
covered with white (1A1), reverse brownish grey
(8E2) (Fig. 3a-f). No growth on PDA at 5°C and 37°C.
Conidiogenous cells produced singly, in pairs, verticillate or in dense irregular clusters on prostratehyphae, at first flask-shaped, tapering into threadlike neck, 3.1-9.2×0.7-2.3 μm. Conidia solitary, oval
to subglobose, 2.4-3.9×1.5-2.3 μm (Fig. 3g-k).
Identification of antimicrobial compounds
In a 500 ml Erlenmeyer flask containing F38 broth,
FKI-9593 produced red pigments. Analyses of
HPLC chromatograms and UV spectra showed that
oosporein (Cole et al., 1974; structure not shown inthis paper) and two different compounds were evident, as detected by UV at 210 nm. Oosporein isolated from the FKI-9593 culture broth was identified
by measurement of 1D NMR and MS spectra.
However, other two compounds were of novel structures by measurement of above instruments. Details
on the novel compounds will be reported elsewhere.
In this study, we clarified that L. aphanocladii in
addition produced two novel compounds (not reported) structurally different from oosporein.

RESULTS

Molecular identification
Only one type of fungus was found to grow fromthe Tengu-no-Mugimeshi sample on all media, but
no growth of bacteria found. A fungal isolate (FKI9593=IFM 64743) was recovered from the sample,
and was classified as a member of the genus
Lecanicillium based on verticillate phialides. FKI9593 also produced a red pigment in PDA. FKI-9593
and Lecanicillium aphanocladii Zare & W. Gams
were grouped together with 99% MP BS, 100% NJ
and ML BS support in multi-gene tree (Fig. 2). With
respect to sequence distances determined by the
MegAlign program from the Lasergene 10 package,the isolate of Lecanicillium had 100% similarity withthe sequences of SSU, LSU and TEF regions of L.
aphanocladii CBS 797.84 (KM283811). From thesemicro-morphological characteristics and the multigene phylogenies, FKI-9593 was identified as L. aph︲
anocladii.

Bioactivity of antimicrobial metabolite
Oosporein showed weak inhibition against Proteus
vulgaris among the 13 microorganisms tested using
the conventional paper disc assay (Table 2).

DISCUSSION

Until now, there have been some reports on
Eubacteria, Archaea, Cyanobacteria andKtedonobacteria isolated from Tengu-no-Mugimeshi(Kawamura, 1916; Ono, 1916; Yagi, 1936; Okada, 1937;
Wang et al., 2019) but only two reports of fungi
being isolated from it (Kawamura, 1916; Ono, 1916).
Among them, a verticillium-like fungus was isolated
from Tengu-no-Mugimeshi from Mt. Kurohime in1902 (Ono, 1916).
Verticillium sect. Prostrata W. Gams (Clavicipitaceae
Kreisel) was divided into at least four distinct clades
based on sequences of the ITS region including the
5.8S rRNA gene (Zare et al., 2000; Gams & Zare,
2001). The genus Lecanicillium W. Gams & Zare
(type: L. lecanii (Zimm.) Zare & W. Gams) was segregated from the former Verticillium sect. Prostrata

Species description
Lecanicillium aphanocladii Zare & W. Gams, Nova
Hedwigia 73: 27, 2001. Fig. 3.
MycoBank no.: MB 484541.
Colonies on PDA 34-36 mm in diameter after 7
days at 25°C, very raised, covered with white (1A1),
reverse violet brown (10E5), produced diffusing reddish-purple pigment. Colonies on MEA 36-37 mm in
─7─The constituent fungi of Tengu-no-Mugimeshi stored for 80 years

Higo et al.

a

c

e

b

d

f

g

i

2μm
Fig. ₃

h

k

j

10μm

5μm

ʟｅｃａｎｉｃｉˡˡｉｕⅿ ａｐʰａｎｏｃˡａｄｉｉ FKI-₉₅₉₃ (=IFM 6₄₇₄₃). a: Colony on PDA at 25℃ for 7 days. b: Reverse sideon PDA. c: Colony on MEA at 25℃ for 7 days. d: Reverse side on MEA. e: Colony on PCA at 25℃ for 7 days.
f: Reverse side on PCA. g, h: Conidiophores. i-k: Conidia, SEM. Bars: g, j 10 μm; h 50 μm; i 2 μm; k 5 μm
─8─Microb. Resour. Syst. June 2021

Vol. 37, No. 1

Table ₂

Antimicrobial activity of Oosporein

Test organisms

Inhibition zone (mm)at 50 μg/disc(8 mm disc)

Inhibition zone (mm)at 10 μg/disc(6 mm disc)Bacillus subtilis ATCC 6633
Staphylococcus aureus ATCC 6538p
Kocuria rizophila ATCC 9341
Mycobacterium smegmatis ATCC 607
Escherichia coli NIHJ
Pseudomonas aeruginosa IFO 3080
Xanthomonas oryzae KB 88
Proteus vulgaris NBRC 3167
Klebsiella pneumoniae ATCC 10031
Candida albicans ATCC 64548
Saccharomyces cerevisiae ATCC 9763
Aspergillus niger ATCC 6275
Mucor racemosus IFO 4581

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besides Simplicillium, Pochonia Bat. & O.M. Fonseca,
Haptocillium W. Gams & Zare, and Rotiferophthora
G.L. Barron, and was described with thirteen taxa
including L. aphanocladii (Gams & Zare, 2001; Zare
& Gams, 2001). It is therefore presumably suggested that this verticillium-like fungus isolated previously from Tengu-no-Mugimeshi could be the same
species as L. aphanocladii identified by us. Only
one fungus was isolated from the specimen of
Tengu-no-Mugimeshi in this study.
Lecanicillium aphanocladii is known as a mainly
mycoparasitic fungus: cobweb and spotting in cultivated Agaricus (Zare & Gams, 2001), parasitizing
uredospores and inducing teliospore formation inrust fungi (Biali et al., 1972; Forrer, 1977; Koç &Défago, 1983; Srivastava et al., 1985; Volker & Boyle,1994) and parasitizing powdery mildew (Podosphaera
fuliginea (Schltdl.) U. Braun & S. Takam.; Heijwegen,1988; Hara et al., 2009). And this species is also
known as an entomopathogenic fungus: mosquito
larvae (López Lastra et al., 1992, 2002); Bombyx mori(Patil et al., 1994).
Moreover, Lecanicillium spp. produce many interesting secondary metabolites (e.g., butoxylspiciferin,(Edrada et al., 2000); spiciferone A, (Hwang et al.,2016); verlamelin and this analogue, (Ishidoh et al.,2014)). Lecanicillium aphanocladii produce a red
pigment in the agar (oosporein, Zare & Gams, 2001).
Thus, the genus Lecanicillium is highly capable ofproducing compounds. In this study, two novelcompounds (not reported) discovered from L. apha︲nocladii FKI-9593.
At least the lid and body of the specimen bottle

were adamantly sealed with the vaseline adhering
to it. The mouth of the bottle was heated with an
alcohol lamp, the vaseline was dissolved, and the lid
was opened for the present study. Since the specimens was not exhibited and had been placed in disuse at the elementary school for a long time, it isprobable that it was well sealed before we opened
them. It is therefore unlikely that the specimen
would have been contaminated by the time we
opened it. From these observations, there are several possible reasons why only L. aphanocladii was
isolated from the specimen of Tengu-no-Mugimeshi
in this study. Firstly, at the time of collection in
1939, we can assume many types of microbes coexisted in Tengu-no-Mugimeshi, but most of the microorganisms died over time due to environmental factors; drying, temperature and nutrient depletions,and only L. aphanocladii survived. Secondly, it
could be that the oosporein produced by L. aphano︲cladii killed the co-existing microbes. For it is
known that some microorganisms are able to takeup iron by making siderophores when there is a
shortage of iron in the environment and are able to
grow. Therefore, we believe that microorganismsthat are unable to make siderophores themselves
may have died during the 80 years of storage. The
researchers confirmed experimentally that L. apha︲nocladii isolates produced oosporein, a type of siderophores. We believe that this is one of the factorsthat kept it alive for 80 years. However, to confirm
any hypothesis it will be necessary to obtain fresh
Tengu-no-Mugimeshi. Collection of a fresh sample
will facilitate an investigation of whether L. aphano︲
─9─The constituent fungi of Tengu-no-Mugimeshi stored for 80 years

cladii actually remains a component in Tengu-noMugimeshi and, if so, what other microorganisms coexist with it.
Of further note, there is anecdotal evidence that
Buddhist monks living in the mountains have eaten
Tengu-no-Mugimeshi for centuries (Yagi, 1936),
although it is unknown whether it is safe to eat.
From our results, if L. aphanocladii was present inTengu-no-Mugimeshi, and oosporein would possibly
be present at unknown concentrations. Oosporein is
known to have lethal effects in young cockerels aswell as having phytotoxic and growth inhibiting
properties (Cole et al., 1974), and there is a reportthat significant damage occurs in the kidney andsplenocytes of oosporein-treated mice (Ramesha etal., 2015). To ascertain if Tengu-no-Mugimeshi issafe to eat, fresh samples and oosporein need to be
assayed, probably against human kidney cells, splenocytes and hepatocytes. In addition, L. aphanocla︲
dii can be cultivated not only in the F38 medium
used this time but also in three other types of media.
However, oosporein was not produced when the fungus was grown on the other media. Consequently, if
the nutrient composition of Tengu-no-Mugimeshi isrelated to the production of oosporein, it may prove
harmful to humans. In order to determine whether
the nutrient composition of Tengu-no-Mugimeshi isrelated to the production of oosporein, it is necessary to investigate what components in the F38
medium are involved in the production of oosporein.

DISCLOSURE

The authors declare no conflicts of interest. All
the experiments undertaken in this study comply
with the laws of Japan.

ACKNOWLEDGEMENTS

We are grateful to Distinguished Emeritus
Professor Satoshi Ōmura (Kitasato University) for his
helpful supports and valuable suggestions. And also,
we would like to thank Mr. Yoshihiko Kubo, Nagano
City, for his assistance in arranging the survey ofTengu-no-Mugimeshi sample for the study. This
study was partly supported by the Platform Projectfor Supporting Drug Discovery and Life Science
Research (Basis for Supporting Innovative Drug
Discovery and Life Science Research (BINDS)) from
AMED, Japan under grant number JP19am0101096.
This research was partially supported by AMED
under grant number JP19ae0101047 as well. This

Higo et al.

research was also partially supported by Researchon Development of New Drugs from AMED under
grant number JP18ak0101053. This study was supported, in part, by the fund from a Kitasato
University Research Grant for Young Researchers.

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The constituent fungi of Tengu-no-Mugimeshi stored for 80 years

Higo et al.

長野県黒姫山の「天狗の麦飯」から分離された糸状菌 Lecanicillium aphanocladii
肥後茉由佳 1），小島裕貴 1），岩月正人 1，2），田辺智隆 3），矢口貴志 4），奥田
1）

徹 5），糟谷大河 6），野中健一 1，2）

北里大学大村智記念研究所，2）北里大学大学院感染制御科学府，3）戸隠地質化石博物館，4）千葉大学真菌医学研究センター，
5）

株式会社ハイファジェネシス，6）慶應義塾大学生物学教室

天狗の麦飯は，中部地方の火山帯，標高の高い地域に点在し，（藍藻類を含む）真正細菌，古細菌，糸状菌等，複数の微生物群からなる微生物の塊である．今回分離源とした天狗の麦飯は，昭和 14 年に黒姫山で採集され，ガラス瓶に封入されて 80
年間長野市内の小学校に保管されていた標本である．本研究では，この天狗の麦飯の標本から分離した糸状菌について報告する．この天狗の麦飯を希釈平板法により 3 種類の培地に塗布し，27℃で 1 週間培養した．その結果，各培地で形態的特徴の同じ 1 種の糸状菌のみが検出され，細菌など他の微生物は一切検出されなかった．この 1 種の糸状菌について，コロニー性状確認，各種顕微鏡による形態観察，3 遺伝子領域に基づく系統解析を行ったところ，Lecanicillium aphanocladii であると同定した．また，本菌株の微生物培養液を用いて 13 種の微生物に対する抗菌試験を行ったところ，Proteus vulgaris NBRC
3167 に対し弱い抗菌活性が確認された．NMR などの各種機器分析により，本活性化合物を oosporein であると同定した．

─ 12 ─