Stimulation of the biosynthesis of the antibiotics lambertellols by the mycoparasitic fungus Lambertella corni-maris under the acidic conditions produced by its host fungus in vitro

Article abstract of DOI:10.1271/bbb.60667

The filamentous fungus, Lambertella corni-maris (L. corni-maris), a mycoparasite on Monilinia fructigena, produces the antibiotics, lambertellols A (1), B (2), and lambertellin (3), in a substantial amounts under acidic conditions, whereas these antibiotics were hardly detected when the fungus was cultured on a potatosucrose (PS) medium without added acids. Our investigations also revealed that the host, M. fructigena, changed its surroundings into acidic conditions, suggesting that the acidic conditions acted as kairomones that stimulated the production of 1-3.

Full text of DOI:10.1271/bbb.60667

Biosci. Biotechnol. Biochem., 71 (5), 1230–1235, 2007
Stimulation of the Biosynthesis of the Antibiotics Lambertellols
by the Mycoparasitic Fungus Lambertella corni-maris under
the Acidic Conditions Produced by Its Host Fungus in Vitro
1
1
1
Takanori MURAKAMI, Noboru TAKADA, Yukio HARADA,
2
1;y
Toshikatsu OKUNO, and Masaru HASHIMOTO
1
Faculty of Agriculture and Life Science, Hirosaki University, 3-Bunkyo-cho, Hirosaki 036-8561, Japan
University of Akita Nursing and Welfare, 2-3-4 Shimizu, Odate 017-0046, Japan
2
Received November 27, 2006; Accepted January 24, 2007; Online Publication, May 7, 2007
[doi:10.1271/bbb.60667]
The filamentous fungus, Lambertella corni-maris
L. corni-maris), a mycoparasite on Monilinia fructige-
interfered with host growth during competitive culture
on a PSA medium, whereas L. corni-maris did not at a
glance.
During an investigation of the chemical substances
responsible for the mycoparasitism of apples, we have
studied the metabolites produced by L. sp. 1346 and
(
na, produces the antibiotics, lambertellols A (1), B (2),
and lambertellin (3), in a substantial amounts under
acidic conditions, whereas these antibiotics were hardly
detected when the fungus was cultured on a potato-
sucrose (PS) medium without added acids. Our inves-
tigations also revealed that the host, M. fructigena,
changed its surroundings into acidic conditions, sug-
gesting that the acidic conditions acted as kairomones
that stimulated the production of 1–3.
4
,5)
found lambertellols A (1) and B (2)
antifungal substances (Fig. 1), together with minor
as the major
6
)
7)
amounts of lambertellol C, neolambertellin, and
8
)
known lambertellin (3). In order to elucidate the
behavioral difference against host M. fructigena be-
tween L. corni-maris and L. sp. 1346, we studied the
relationship between the secondary metabolites of these
fungi and the culture conditions.
Key words: lambertellols; pH-dependent production;
mycoparasite
Our investigations disclosed that L. corni-maris could
produce 1, 2 and 3 in large amounts by culturing it under
acidic conditions. The PSA medium around the hyphal
surroundings of host M. fructigena was found to become
acidic. This indicated that the acidic conditions around
the host hyphae stimulated L. corni-maris to initiate
biosynthesis of these antifungal substances. Since the
acidic region was limited only to that where the host
hyphae were observed, L. corni-maris did not produce
these compounds in a competitive culture on PSA until
its hyphae had developed to reach the host.
Hori reported in 1912 the discomycete, Seclerotinia
phaeospora Hori, in apples as a causal fungus of fruit
rot, the so-called ‘‘Natsu-Nenju’’ disease in Japanese.
1
)
Based on his bionomical observations, he mentioned
that this fungus had two stages in its lifecycle:
anamorphic (asexual) and teleomorphic (sexual). The
fungus was then raised to a new genus and species,
2
)
Phaeosclerotinia nipponia Hori, in 1916.
One of the present authors (Y.H.) reported in 1990
that Phaeosclerotinia nipponica Hori is a misapplied
name in reality, and that Hori’s observation can be
3
)
explained by a mycoparasitic development of Lamber-
tella corni-maris (L. corni-maris) on Monilinia fructi-
gena (M. fructigena), which is a common pathogenic
fungus in apples in Japan. Simultaneous culturing of
L. corni-maris and M. fructigena on a potato-sucrose-
agar (PSA) medium for a couple of months resulted only
in L. corni-maris being isolated, even from the region
where M. fructigena had developed independently.
Harada has recently identified Lambertella sp. 1346
Materials and Methods
The discomycetes used for our experiments were
isolated by one of the present authors (Y.H.). Lamber-
tella corni-maris, L. sp. 1346, and M. fructigena were
collected in Aomori prefecture in Japan in 1988, 1989,
and 2003, respectively. They were stored on a PSA
medium (ca. 2-mm thickness) in 9-cmꢀ Petri dishes at
ꢀ
20 C. The PSA medium was prepared from sucrose
(20 g), an aqueous potato extract (1.0-liter, 200 g from
potato), and agar (20 g).
(L. sp. 1346) as a novel and similar mycoparasite on
M. fructigena. Under similar conditions, L. sp. 1346
y
To whom correspondence should be addressed. Tel/Fax: +81-172-39-3782; E-mail: hmasaru@cc.hirosaki-u.ac.jp
Abbreviations: PSA, potato-sucrose agar; PS, potato-sucrose; ODS, octadecyl silyl

Production of Lambertellols under Acidic Conditions
1231
O
OH
O
OH
O
O
OH
O
O
O
H C
O
3
H C
3
O
OH
OH
O
H C
3
Lambertellol A (1)
Lambertellol B (2)
Lambertellin (3)
Fig. 1. Structures of the Secondary Metabolites Isolated from Lambertella sp. 1346.
Lambertellols A (1) and B (2) were prepared by fol-
4
30% aqueous CH3CN (150 ml), and 10 ml of the solution
was injected into the HPLC instrument. Compounds 1–3
were detected by their fluorescence at 550 nm, with
excitation at 350 nm and UV 430 nm, as described in the
text.
,5)
lowing our established procedure. The HPLC analysis
ꢀ
was performed with a Merck LiChrospher 100 RP-18e
5
-mm column (250 ꢁ 4:0 mm I.D.) and a Waters 626
pump equipped with a Waters 600S controller, Waters
74 scanning fluorescent detector, and 996 photodiode
4
1
array detector. The H-NMR data for 1 and 2 thus
)
Cultivation of L. corni-maris in the presence of
Monilinia broth. The Monilinia broth was prepared by
culturing M. fructigena in a potato-sucrose (PS) medium
4
obtained were identical to those reported.
Lambertellin (3) was prepared by chemical conver-
4
)
ꢀ
sion from lambertellols. A solution of a mixture of 1
and 2 (20 mg, 1:2 = 2:3 at UV 330 nm) in a mixture of
MeOH (10 ml) and H2O (10 ml) was allowed to stand in
the ambient atmosphere at room temperature for 1 week.
The dark reddish precipitate was collected by filtration,
followed by recrystallization from acetone to give
(200 ml) for 14 days without shaking at 25 C, and then
filtering. Lambertella corni-maris was seeded on another
PS medium (150 ml baffled flasks), and cultured for
ꢀ
7 days at 25 C on a rotary shaker (110 rpm). Into
the medium, the Monilinia broth (100 ml) was added
through a cotton filter, and then resulting mixture was
further cultured under the same conditions for an addi-
tional 7 days. The supernatant of the culture medium
was directly analyzed by HPLC. This series of experi-
ments was triplicated, and the mean amounts of 1–3 are
used in the discussion.
1
pure lambertellin (3, 12 mg). The H-NMR data were
8
)
identical to those reported.
Moniliniol and 3-Cl moniliniol9) were isolated from
the culture broth of M. fructigena after culturing in the
ꢀ
PS medium (200 ml ꢁ 20 flasks) at 26 C for 2 weeks
without shaking. The broth after filtration was extracted
with EtOAc (2.0-liter). The extract was concentrated
under reduced pressure to give a residue. Silica gel
column chromatography of this residue using hexane–
acetone (80:20) as the eluent afforded moniliniol and 3-
Cl moniliniol. The structures of these compounds were
Cultivation of L. spp. under acidic conditions. The PS
medium (200 ml) was adjusted to pH 3.8 by adding a
1.0 M citric acid solution (ca. 1.0 ml). After sterilization
by autoclaving, Lambertella species were seeded and
cultured under the same conditions as these already
mentioned. After 8 days, the supernatant (1.0 ml) was
collected and directly analyzed by HPLC. Experiments
using L. corni-maris with hydrochloric acid instead of
citric acid were also performed. This series of experi-
ments was duplicated, and the means of the data are used
in the discussions.
9
)
fully assigned by comparing with the reported data.
Competitive culture of Lambertella species with the
host M. fructigena. The parasite, L. sp. 1346, and the
host, M. fructigena, were seeded on a freshly prepared
PSA medium in a Petri dish (9 cmꢀ), leaving ca. 4 cm of
ꢀ
space. The Petri dish was kept at 25 C for two weeks
(Fig. 2, left). A similar experiment was performed using
L. corni-maris used instead of L. sp. 1346 (Fig. 2, right).
Cultivation of L. spp. under neutral conditions.
Sodium citrate dehydrate (290 mg) was added when
the PS medium (200 ml) was prepared. Lambertella
corni-maris was cultured in this medium under the same
condition as those just mentioned. After culturing for 8
days, the supernatant was sampled and analyzed by
HPLC. A similar experiment using L. sp. 1346 was also
performed. This series of experiments was duplicated,
and the means of the data are used in the discussions.
Detection of lambertellols from L. corni-maris on the
PSA medium by culturing in the presence of M. fructi-
gena. Both L. corni-maris and M. fructigena were
seeded on a Petri dish (9-cmꢀ), leaving ca. 6 cm of
ꢀ
space. After cultivating at 20 C for 18 days, five
sections (8-mmꢀ, the points where L. corni-maris and
M. fructigena were transplanted as well as three equi-
distant points) were sampled with a cork borer. Each
section was put in CH3CN (3.0 ml). After 12 h, these
were independently filtered through cotton pads and
concentrated in vacuo. Each residue was diluted with
Results and Discussion
Lambertella corni-maris is the initially identified
mycoparasite on M. fructigena. In contrast with L. sp.

1232
T. MURAKAMI et al.
M. fructigena
L.sp.1346
M. fructigena
L. corni-maris
Fig. 2. Competitive Cultures of the Mycoparasite Lambertella spp. with the Host Monilinia fructigena on PSA Medium.
1
346, L. corni-maris did not suppress the host M. fruc-
tigena at a glance under competitive culture on PSA
medium as shown in Fig. 2. However, only L. corni-
maris was isolated from the medium with L. corni-maris
and M. fructigena after storing for a couple of months.
Our preliminary experiments showed that L. corni-maris
produced 1–3, but at quite low levels (near the detection
limit) under the same conditions used for L. sp. 1346
which provided several milligrams of the antibiotics
from 4-liter of the culture broth.
We assumed that L. corni-maris also produced 1–3,
but this production was limited only in the presence of
the host. Based on this assumption, we investigated the
influence of this production by L. corni-maris on the
host metabolites.
Prior to these experiments, we developed sensitive
and specific analytical conditions for identifying 1–3.
Lambertellin (3; ꢁmax 430 nm, " 4800) could be iden-
tified by HPLC at UV 430 nm. The limit of detection
was estimated to be 100 ng/injection. This condition
was specific enough for quantifying 3 from a crude
extract. However, the conditions were much less sen-
sitive for 1 and 2. The ꢁmax values of 1 and 2 were
both 261 nm (" 9500 for both), but this wavelength was
applicable only for pure samples. The crude extract gave
many signals due to the impurities involved, which
decreased the signal-to-noise ratio. We found that these
compounds could be specifically quantified by fluores-
cence at 550 nm (excitation at 350 nm), which sensed
around 100 ng/injection for both. These methods en-
abled us to analyze 1–3 directly from the PS medium.
When L. corni-maris and M. fructigena were com-
petitively cultured for 18 days on the PSA medium, the
fungi were intermixed and three zones were found by
visual observation: one solely inhabited by the parasite,
one solely inhabited by the host, and another one
inhabited simultaneously by both the parasite and host
zone
(µg/cm )
(µg/cm )
(µg/cm )
I
II
III
IV
V
2
1
2
3
n.d.
n.d.
n.d.
n.d.
n.d.
0.39
0.41
1.74
4.36
0.22
0.41
0.31
n.d.
n.d.
n.d.
2
2
n.d., not detectable (less than limit of detection)
Fig. 3. Production of Lambertellol A (1), Lambertellol B (2), and
Lambertellin (3) by L. corni-maris in the Presence of the Host
M. fructigena.
and M. fructigena (sections III and IV). In contrast, 1
and 2 were below the detection level in sections I, II and
V. In these regions, the parasite or the host existed
independently. Lambertellin (3) was observed in region
II, but the amount was quite small. These results may
support our assumption that L. corni-maris exuded these
antibiotics by detecting the host M. fructigena. Two
explanations are possible for this phenomenon: chemical
stimulation so-called allelopathy and inducement by
physical contact.
(
Fig. 3). Five small sections (8 mmꢀ) were sampled
from the medium, extracted with acetonitrile, and
analyzed by HPLC under the conditions described.
Interestingly, 1–3 were detected in considerable
amounts in the regions inhabited by both L. corni-maris
These possibilities were investigated by culturing
L. corni-maris in a liquid medum in the presence of
the host M. fructigena culture broth. It was found that
L. corni-maris provided 1 (18 mg/ml), 2 (52 mg/ml), and

Production of Lambertellols under Acidic Conditions
1233
L. corni-maris
L. sp. 1346
Culture for 7 days
Culture for 7 days
Culture broth of
M. fructigena
Blank
PS medium
C
Culture for additional 7 days
A
B
2
1
A
B
C
0
.0
5.0
10.0
15.0
20.0 min
Fig. 4. HPLC Chromatograms of the Culture Broth of L. corni-maris with the M. fructigena Culture Broth (A), with the Blank PS Medium (B), and
with the Culture Broth of L. corni-maris Obtained under the Usual Conditions (C).
Lambertellin (3) was not detected under these conditions.
3
(9.0 mg/ml) as shown in Fig. 4 (chromatogram A,
amounts without a Monilinia extract after culturing for 8
days (Table 1, run ii).* Under regular conditions, the
compounds were hardly found (run i). These results may
indicate citric acid to be responsible for this; sodium
citrate, however, did not increase the production of 1–3
(run iii). We found HCl also induced this productions
(run iv), which suggests acidic conditions were respon-
sible.
chromatograms at UV 430 nm detecting 3 are not
shown) when it was cultured with the host broth for 7
days. These compounds were within the detection limits
(
chromatogram B: 1, <0:1 mg/ml, 2, <0:1 mg/ml, 3,
.9 mg/ml) in the control experiment for which the blank
PS medium was added instead of the culture broth. In
0
these experiments, L. corni-maris had been cultured for
1
0 days before adding the host broth or the blank PS
medium. Notably, L. sp.1346 provided 1 (44 mg/ml) and
(75 mg/ml) after 7 days of culture without any addition
In contrast, L. sp. 1346 produced considerable
amounts of 1–3 under all the conditions examined (runs
v–vii). The addition of either citric acid or sodium
citrate seemed to decrease the levels of 1–3 compared
with the regular condition. This may have been due to
the slightly lower growth rate of L. sp. 1346 under these
conditions.
We found that L. sp. 1346 also dramatically decreased
the pH value of the culture medium. Neutral conditions
could not be maintained even with sodium citrate (run
vii). After culturing for a week, the pH value of the
culture medium with sodium citrate became 3.5, where-
as the initial pH value was 7.5. In contrast, L. corni-
maris did not markedly affect the pH value of the
medium. These results suggest that L. sp. 1346 stimu-
lated the production of 1–3 by acidifying the environ-
ment by itself.
2
of the host broth (chromatogram C). Lambertellin (3)
was also found (0.3 mg/ml) in this sample, but in a
relatively low quantity. These experiments proved that
chemical conditions stimulated L. corni-maris to pro-
duce these antibiotics, and not physical contact.
However, all our attempts to fractionate the active
substance(s) failed. The activity could not be extracted
by organic solvents. Fractionation by ODS was not
reproducible. Neither of the major secondary metabo-
lites of M. fructigena, moniliniol and 3-Cl moniliniol,
induced the biosyntheses of 1–3.
Interestingly, the PS medium was found to become
strongly acidic (pH 4.2) by culturing the host M. fructi-
gena for a week. The original pH value was 6.3 before
the culture. This result suggested that the acidic
conditions might be the stimulant. To confirm this
assumption, we cultured L. corni-maris in a liquid
medium under acidic conditions. When the pH value
of the medium was adjusted to 3.8 with citric acid before
seeding, L. corni-maris afforded 1–3 in considerable
*
Our preliminary experiments revealed that the production of 1–3
was synchronized with the logarithmic growth phase of the fungi,
especially for L. corni-maris. Since acidic conditions slightly
decreased the growth rates of both L. corni-maris and L. sp. 1346,
HPLC analyses were performed after culturing for 8 days in these
experiments.

1234
T. MURAKAMI et al.
Table 1. Production of Lambertellol A (1), Lambertellol B (2), and Lambertellin (3) by L. corni-maris and L. sp.1346 after Culturing for 8 Days on
a PSA Medium under Various Conditions
did not produce 1–3 independently on PSA, the host
could grow freely until its hyphae reached the parasite.
On reaching the host, L. corni-maris would then begin
to produce these antibiotics to deteriorate the host.**
This might be the reason why only L. corni-maris was
isolated after simultaneous cultivation with M. fructige-
na. On the other hand, L. sp. 1346 was found to
markedly acidify the environment. This fungus might
stimulate itself to this production by acidifying its
environment.
Acknowledgment
Part of this work was supported by grant-aid for
scientific research (No. 17580090) from the Ministry of
Education, Culture, Sports, Science and Technology of
Fig. 5. pH Test Strip with M. fructigena on the PSA Medium.
Japan. We also thank Suntory Institute for Bioorganic
X, the point M. fructigena was seeded.
Research for the SUNBOR Grant.
The decrease in pH value by the host M. fructigena
was also observed on PSA gel. Since it is difficult to
identify the acidity of gels by pH, we used a pH test strip
as the indicator. When the pH test strip was placed on
the PSA culture medium, only the area with the host
M. fructigena was found to be acidic, while the region
without M. fructigena was neutral as shown in Fig. 5.
This result indicates that the spreading rate of the acidic
region was almost identical to the growth rate of the host
hyphae on the PSA medium.
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)
)
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**
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