183

Mycoscience 35:183-186, 1994

Short Communication

Identification of Penicillium sp, NR 6 5 6 4 and taxonomic

notes on P. janthinellum

Shoko M a t s u k u m a , Yuri Nishizuka, Junko W a t a n a b e and Toru Okuda

Department of Microbiology and Taxonomy, Nippon Roche Research Center, 200 Kajiwara, Kamakura 247, Japan

Accepted for publication 1 April 1994

The fungal strain NR 6564, which produces a new series of antifungal antibiotics, Ro 0 9 - 1 4 7 0 and its congeners, was
identified as Penicilliumjanthinellum from its cultural and morphological properties. The identification was confirmed by
analysis of the ubiquinone system and DNA-DNA hybridization.

Key Words DNA-DNA hybridization; Penicillium janthinellum; P. simplicissimum; ubiquinone.

Penicillium sp. NR 6564, a strain producing a new series ly ellipsoidal, sometimes pyriform with apiculate ends,
of antifungal antibiotics comprising Ro 09-1470 and its with smooth to finely roughened walls (Fig. 1).
congeners, was isolated from a soil sample collected in Although there were considerable variations in penicillus-
Hong Kong in 1987 by a serial dilution method on a type, phialide neck length, and conidial shape and surface
modified malt extract agar (Matsukuma et al., 1992). ornamentation among the strains examined, NR 6564
Ro 09-1470 is the first natural antifungal compound to in- most closely resembled IMI 40238. In addition, the mor-
hibit the P450 lanosterol C-14 demethylase of fungi phological and cultural characteristics of NR 6564 clearly
(Aoki et al., 1992). differed from those of P. simplicissimum (data not
We examined cultural and morphological properties shown).
of NR 6564 according to Pitt (1979, 1988), in compari- As described above, NR 6564 showed closer resem-
son with several reference strains. The reference strains blance to Penicillium janthinellum IMI 40238 and IMI
used were P. janthinellum Biourge IMI 40238, IMI 71625 than to the other references strains. However,
71625, FRR 1893, FRR 1962, FRR 513, IMI 68213 and discrepancies were considerable from strain to strain in
P. simplicissimum (Oudem.) Thorn IMI 40032, IMI this species. Pitt (1979, 1988) described P. janthinel-
166578, FRR 1859, FRR 89, FRR 725. These strains lure as being one of the Penicillium species that is difficult
were obtained from the following culture collections: to define, because it shows great variation. The nomen-
ATCC (American Type Culture Collection, Rockville, MD, clature treatment is also confusing. Penicilliumjanthinel-
U.S.A), FRR (CSIRO, Division of Food Processing, North lure has been treated as a synonym for P. sirnplicissimum
Ryde, N.S.W., Australia), and IMI (International Mycologi- (Oudem.) Thorn (Stolk and Samson, 1983; Fassatiov~
cal Institute, Egham, Surrey, U.K.). and Kubatov& 1990; Frisvad and Filtenborg, 1990).
Table 1 shows the cultural properties of strain NR
6564 and six strains of P. janthinellum. After we had re-
vived the sporulation ability of IMI 40238 (Nishizuka et
al., 1993), we found that NR 6564 formed characteristic
colonies on CYA and MEA, dull red soluble pigments, dar-
ker reverse side color, and no germination of conidia at
5°C on CYA, indicating a closer afffinity to IMI 40238 (a
strain derived from the neotype) than to the other refer-
ence strains.
Table 2 shows the morphological properties of NR
6564 and six other strains of P. janthinellum. NR 6564
characteristically formed smooth, thin-walled stipes.
The penicilli were biverticillate. The metulae were most-
ly divergent but sometimes compact and parallel. The
phialides were ampulliform, gradually tapering to collula
that were usually long and slender but occasionally very
short. The conidia were most often subglobose to broad- Fig. 1. Penicilliurn sp. NR 6564 (Bar: lOpm).
184 S. Matsukuma et al.

Table 1. Cultural properties of Penicillium sp. NR 6 5 6 4 and P, janthinellum.

Penicillium sp. Penicillium janthinellum

NR 6564 IMI 40238 IMI 71625 FRR 1893 FRR 1962 FRR 513 IMI 68213

CYA 2 5 ° C ~ 39-43 25-26 18-20 34-37 35-37 23-29 22-26

MEA 25°C ~ 43-45 38-42 27-30 39-41 49-50 60-62 31-37
G25N 25°C ~ 7-11 10-11 7-9 16-17 17-18 14-15 12-13
5°C on CYA ~ 0 0 0 7 5-6 germination germination
37°C on CYA ~ germination 12-13 germination 12-13 10-13 4-5 13
CYA
Conidial mass light graysh not observed pale blue green pale bluish pate blue green not observed grayish green
color green green
Mycelial color pale pink white-cream white white white pale yellowish white
brown
Reverse color dard reddish dark yellow brownish olive light yellow yellowish dull yellowish yellowish gray
brown brown orange
Soluble pigment dull red none ~ none ~ none none none none
MEA
Conidial mass pale blue green pale blue green poor greenish gray grayish green pale blue green pale green
color
Mycelial color white- white white white white white white
pale yellow
Reverse color brownish olive dull yellow dull yellow pale yellowish yellowish white pale yellow yellowish white
green
Soluble pigment none none none none none none none

Colony diam are expressed in mm after 7 days. ~ Dull red pigmentation appeared after 14 days.

We, t h e r e f o r e , applied c h e m o t a x o n o m i c and m o l e c u - 6 5 6 4 belongs t o t h e s u b g e n u s Furcatum, b e c a u s e it c o n -

lar t a x o n o m i c m e t h o d s t o s u p p o r t o u r i d e n t i f i c a t i o n .
U b i q u i n o n e analysis by h i g h - p e r f o r m a n c e liquid chro-
m a t o g r a p h y w a s carried o u t by t h e m e t h o d of Kuraishi et
al. ( 1 9 8 5 ) . In t h e u b i q u i n o n e s y s t e m , Kuraishi et al.
(1991 ) r e p o r t t h a t a l m o s t all t h e species b e l o n g i n g t o t h e
s u b g e n u s Furcatum h a v e Q-9, w h e r e a s t h e species be-
l o n g i n g t o t h e s u b g e n u s Biverticillium, e x c e p t for t h e sec-
t i o n Coremigena, h a v e Q-IO(H2). We c o n f i r m e d t h a t NR
t a i n e d Q-9 as a m a j o r u b i q u i n o n e t y p e .
DNA-DNA hybridization was performed by the
m i c r o t i t e r plate m e t h o d a c c o r d i n g t o Ezaki et al. ( 1 9 8 9 ) .
The h y b r i d i z a t i o n t e m p e r a t u r e w a s d e t e r m i n e d as 4 5 ° C
based on t h e Tm. T m v a l u e s o f t h e D N A s purified f r o m
NR 6 5 6 4 , P. janthinellum FRR 1 8 9 3 , IMI 6 8 2 1 3 , and P.
simplicissimum FRR 7 2 5 w e r e 9 0 ° C , 9 0 ° C , 8 8 ° C , and
89°C, respectively.

Table 2. Morphological properties of Penicillium sp. NR 6564 and P. janthinellum,

Penicillum sp. Penicillium janthinellum

NR 6564
IMI 40238 IMI 71625 FRR 1893 FRR 1962 FRR 513 IMI 68213

Penicillus biverticillate to monoverticillate monoverticillate monoverticillate monoverticillate monover- monoverticilla-

irregular to irregular to irregular to irregular to irregular ticillate re, rarely
biverticillate biverticillate biverticillate biverticitlate biverticillate irregular
with a few with a few with a few with a few biverticillate
metulae metulae metulae metulae with a few
metulae
Phialide
Size (~m) 10.0-16.5x 8.5-11.0× 7.5-9.0× 7,0-9.5 x 6.5-10.0x 8.0-12,0x 7.0-11.0×
2,5-4.0 2.0-3,0 2.0-3.0 2.0-3.0 2,0-2.5 2.5-3,0 2.5-3,5
Shape ampulliform ampulliform ampulliform ampulliform ampulliform amputliform ampulliform
Neck long long long short short long long
Wall smooth smooth smooth smooth smooth smooth smooth
Conidium
Size (/~m) 3.0-4.0 x 3.0-4.0 x 2.5-3.0 × 3.0-3.5 x 2.5-3.0 x 3.0-4.0 x 3.0-4.0 x
2,5-3.0 2.5-3.5 2.5-3,0 3.0-3.5 2.5-3.0 2.5-3.5 3.0-3.5
Shape ellipsoidal with ellipsoidal with ellipsoidal with spherical oval ellipsoidal spherical
apiculate ends apiculate ends apiculate ends
Wall smooth smooth smooth rough smooth spinose spinose
 Identification of Penicillium sp. and taxonomic notes on P, janthinettum 185

Table 3. DNA relatedness between Peniciltium sp. NR 6564 and P. janthinellum.

% Homology with photobiotin labeled DNA

Species Strains
NR6564 IMI40238 IMI71625 FRR 1893 FRR 1962 FRR 513 IMI68213
Penicillium sp. NR 6564 100
P. janthinellum IMI40238 84 100
P. janthinellum IMI71625 64 75 100
P. janthinellum FRR 1893 26 28 32 100
P. janthinellum FRR 1962 31 33 30 53 100
P. janthinellurn FRR 513 11 13 12 9 13 100
P. janthinellum IMI68213 22 34 29 33 35 14 100

Table 4. DNA relatedness between Penicitlium sp. NR 6564 and P. simplicissimum.

% Homology with photobiotin labeled DNA

Species Strains
NR6564 IMI40032 IMI166578 FRR 1859 FRR89 FRR725
Penicillium sp. NR 6564 1O0
P. simplicissimum IMI 40032 34 100
P. simplicissimum IMI 166578 47 47 100
P, simplicissimum FRR 1859 25 36 38 100
P. simplicissimum FRR 89 29 28 34 88 100
P. simplicissimum FRR 725 17 5 17 28 24 100

Table 3 and 4 s h o w the DNA-DNA relatendness be-

t w e e n Penicillium sp. NR 6 5 6 4 and P, janthinellum, and
b e t w e e n Penicillium sp. NR 6 5 6 4 and P, simplicissimum,
respectively. NR 6 5 6 4 s h o w e d high h o m o l o g y w i t h P.
janthinellum IMI 4 0 2 3 8 and IMI 71 6 2 5 but l o w h o m o l o g y
w i t h the other strains of P. janthinellum and all e x a m i n e d
strains of P. simplicissimum.
Therefore, the results obtained support our identifica-
tion by c h e m o t a x o n o m i c a l and molecular t a x o n o m i c a l
methods.
Some strains, h o w e v e r , s h o w e d l o w DNA-DNA
h o m o l o g y values w i t h i n species, indicating t h a t the spe-
cies of P. janthinellum also varied w i d e l y at the genetic
level. A l t h o u g h DNA-DNA relatedness by hybridization
is considered a reliable m e t h o d in bacterial t a x o n o m y , it
has not been so frequently used in the t a x o n o m y of deu-
t e r o m y c e t e s . It has been speculated t h a t the w i d e r spe-
cies c o n c e p t in some d e u t e r o m y c e t e s w o u l d make such
c o m p a r i s o n s difficult. As more data on molecular tax-
o n o m y are a c c u m u l a t e d , such difficulties should be
resolved. From our studies, h o w e v e r , it is clear t h a t
c h e m o t a x o n o m y and molecular t a x o n o m y provide effec-
tive tools for identifying deteriorated or poorly sporulated
strains or for c o m p a r i n g similar strains.
Acknowledgements We thank Dr. Pitt of the CSIRO for kind-
ly allowing us to use his strains. We also thank Dr. Kuraishi and
Prof. Sugiyama of the Institute of Molecular and Cellular
Biosciences, University of Tokyo, for their kind instruction and
for analyzing ubiquinone by HPLC.
We also thank Prof. Tubaki of Nippon University for his kind
suggestions.
Literature cited
Aoki, Y., Yamazaki, T., Kondoh, M., Sudoh, Y., Nakayama, N.,
Sekine, H., Shimada, H, and Arisawa, M, 1992, A new
series of natural antifungals that inhibit P450 lanosterol C-
14 demethylase II. J. Antibiotics 45: 160-170.
Ezaki, T., Hashimoto, Y. and Yabuuchi, E. 1989. Fluorometric
deoxyribonucleic acid-deoxyribonucleic acid hybridization in
microdilution wells as an alternative to membrane filter
hybridization in which radioisotopes are used to determine
genetic relatedness among bacterial strains. Int. J. Syst.
Bacteriol. 39: 224-229.
Fassatiov~i, O. and Kubatov~, A. 1990. Evaluation of the diag-
nostic features of some species of Penicillium section
Divaricatum. In: "Modern concepts in Penicillium and
Aspergillus classification," (ed. by Samson, R.A. and Pitt,
J.I.), pp. 149-157. Plenum Press, New York.
Frisvad, J. C. and Filtenborg, O. 1990. Revision of Penicillium
subgenus Furcatum based on secondary metabolites and
conventional characters. In: "Modern concepts in Penicilli-
um and Aspergillus classification," (ed. by Samson, R.A.
and Pitt, J. I.), pp. 159-172. Plenum Press, New York.
Kuraishi, H., Aoki, M., Itoh, M., Katayama, Y., Sugiyama, J. and
Pitt, J. 1. 1991. Distribution of ubiquinones in Penicillium
and related genera. Mycol. Res. 9 5 : 7 0 5 - 7 1 1 .
Kuraishi, H., Katayama-Fujimura, Y,, Sugiyama, J. and Yokoya-
ma, T. 1985. Ubiquinone systems in fungi I. Trans.
Mycol. Soc. Japan 24: 383-395.
Matsukuma, S., Ohtsuka, T., Kotaki, H., Shirai, H., Sano, T.,
Watanabe, K., Nakayama, N., Itezono, Y., Fujiu, M., Shim-
ma, N., Yokose, K. and Okuda, T. 1992. A new series of
natural antifungals that inhibit P450 lanosterol C-14 de-
methylase I. J. Antibiotics 45: 151-159.
Nishizuka, Y., Fujimori, F., Matsukuma, S. and Okuda, T.
186 S. Matsukuma et al.

1993. Cultivation in soil and freeze-drying revived the
sporulation ability of Peniciltium janthinellum IMI 4 0 2 3 8 .
Bull. JFCC 9: 1 3 5 - 1 3 9 .
Pitt, J. I. 1979. "The genus Penicillium and its Teleomorphic
States, Eupenicilliurn and Taralomyces," Academic Press,
London. 6 3 4 p.
Pitt, J. 1.1988. "A laboratory guide to common Penicillium spe-
cies, 2nd ed.," C o m m o n w e a l t h Scientific and Industrial
Research Organization, Australia. 187 p.
Stolk, A. C. and Samson, R.A. 1983. The ascomycete genus
Eupenicillium and related Penicillium anamorphs. Stud.
Mycol. 23: 1 - 1 4 9 .