A new bioassay technique combining leaf disk and softagar

over-layer methods was developed to investigate the

allelopathic effect of deciduous leaf litters on the growth of

cyanobacteria (

Microcystis aeruginosa

Kütz.). Bioactive substances

exuded from leaf disks caused inhibitory plaques on

the agar plate containing cyanobacteria

,

and the rate of diffusion

depended on the specific leaf disk area. Most of the

leaf litters collected around reservoirs in Japan showed inhibitory

activity to

M. aeruginosa

, with

Rhus trichocarpa

Miq.,

Quercus variabilis

Blume and

Mallotus japonicus

(Thunb.)

Muell. Arg. being the strongest among the 22 tested species.(PDF has 4 pages.

Fujii, Yosiharu

Matsuyama, Minoru

Takamura, Noriko

Tsuda, Kumiko

English

Aquatic Commons

 J. Aquat. Plant Manage.
 
 43: 2005. 43
 
J. Aquat. Plant Manage.
 
 43: 43-46
Assessment Method for Leaf Litters
Allelopathic Effect on Cyanobacteria
 
KUMIKO TSUDA
 
1,4
 
, NORIKO TAKAMURA
 
2
 
, MINORU MATSUYAMA
 
3
 
 AND YOSIHARU FUJII
 
1,5
 
ABSTRACT
 
A new bioassay technique combining leaf disk and soft-
agar over-layer methods was developed to investigate the
allelopathic effect of deciduous leaf litters on the growth of
cyanobacteria (
 
Microcystis aeruginosa 
 
Kütz.). Bioactive sub-
stances exuded from leaf disks caused inhibitory plaques on
the agar plate containing cyanobacteria
 
,
 
 and the rate of dif-
fusion depended on the specific leaf disk area. Most of the
leaf litters collected around reservoirs in Japan showed in-
hibitory activity to 
 
M. aeruginosa
 
, with 
 
Rhus trichocarpa
 
 Miq.,
 
Quercus variabilis
 
 Blume and 
 
Mallotus japonicus
 
 (Thunb.)
Muell. Arg. being the strongest among the 22 tested species.
 
Key words:
 
 allelopathy, bioassay; leaf disk, 
 
Microcystis aerugi-
nosa
 
.
 
INTRODUCTION
 
Severe outbreaks of cyanobacteria (blue-green algae) in
shallow lakes and reservoirs, called “water blooms”, cause se-
rious problems with regard to effective utilization of water re-
sources. 
 
Microcystis
 
 is often associated with the formation of
hepatotoxic peptides such as microcystin (Carmichael 1992).
The presence of high concentrations of microcystin can
bring about serious problems in public water supplies. Some
techniques for suppressing the occurrence of water blooms
have been introduced in the past, such as cultivating plants
with high nutrient absorption characteristics (i.e., 
 
Eichhornia
crassipes
 
 (Mart.) Solms-Laub.) in a lake (Otsuki et al. 1990),
and cutting off the supply of nutrients like nitrogen (N),
phosphorus (P), and iron (Fe) which are key factors to the
growth of cyanobacteria (Sakamoto 1966, Welch et al. 1992).
However, high nutrient concentrations in lakes do not totally
explain the occurrence of water blooms (Moss 1998). For ex-
ample, in some shallow lakes, large macrophyte populations
can suppress water blooms in spite of eutrophication (Schef-
fer et al. 2001, Takamura et al. 2003). Based on this observa-
tion, other important factors are inferred to play an active
role in the competition between macrophytes and cyanobac-
teria. One explanation for this phenomenon is the release of
chemical compounds by these plants that inhibit algal
growth (Morris et al. 2003). These bioactive substances are
called allelochemicals, and the mechanism involved is called
allelopathy (Molisch 1937).
Allelopathy has been reported in littoral areas (Gross
1999), and in the last decade, many studies have demonstrat-
ed the algicidal effect of leaf litter and straw (Newman and
Barrett 1993, Ridge et al. 1999). A method to assess allelo-
pathic effects of leaf litters to higher plants has been estab-
lished (Fujii et al. 2004), however, no method to determine
the effect of leaf litters on cyanobacteria has been reported.
This study evaluates the allelopathic effect of deciduous leaf
litters on bloom-forming cyanobacteria by combining the
soft-agar over-layer technique (Uchida et al. 1998, Yamamoto
1978) and leaf disk method.
 
MATERIALS AND METHODS
 
The algal strain (NIES-88) 
 
Microcystis aeruginosa
 
 used in
this bioassay was obtained from the microbial collection of
the National Institute for Environmental Studies (NIES), Ja-
pan. It was pre-cultured in sterilized CT medium (Watanabe
and Ichimura 1977) for 15 d under the following conditions:
a photosynthetic photon flux density of 66.6 µmol photons/
m
 
2
 
/s, 12:12 h light: dark photoperiod, and 30:25 C, respec-
tively. The algae were collected by centrifugation at 3000 g
for 30 min.
CT medium (10 ml) with agar (0.75% w/v) was poured
onto petri dishes (90 mm in diameter) and left to gelatinize
(bottom layer). CT medium (3 ml) with agar (1.0% w/v) at
40 to 50 C was mixed well with 3 ml of the concentrated al-
gae suspension. This algae-mixed medium was then spread
onto the gelatinized bottom layer, and left to harden (top
layer).
Deciduous leaf litters for 22 species collected around the
reservoirs at South Hyogo Prefecture (34N-134E) were
drilled with a leaf disk punch (Fujiwara Scientific Co., To-
kyo, Japan) and sterilized with ultraviolet irradiation (257.3
nm, 6 h for each faces). Paper disks (ADVANTEC, Japan)
sterilized with heat (150 C, 6 h) were used for control. Both
leaf and control disks were placed over the top layer of the
agar plates; a total of two disks were on a plate. These plates
were then incubated under the same pre-culturing condi-
tions for 7 d. A schematic diagram of this method is shown
in Figure 1. The degree of algae growth inhibition was deter-
mined by both size and characteristics of the plaque zones
formed around the leaf disk. All experiments were run in
quadruplicate.
 
1
 
National Institute for Agro-Environmental Science, Chemical Ecology
Unit, 3-1-3 Kan-non-dai, Tsukuba 305-8604, Japan.
 
2
 
National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba
305-8506, Japan.
 
3
 
Hyogo Prefectural Technology Center for Agriculture, Forestry and
Fisheries, 1533 Minamino-oka-kou, Befu-cyo, Kasai 679-0198, Japan.
 
4
 
Present address: Laboratory of Soil Science, Department of Biological
and Environmental Society, Faculty of Agriculture, Kobe University, 1-1
Rokko-dai, Kobe, Hyogo 657-8501, Japan.
 
5
 
e-mail: yfujii@affrc.go.jp. Received for publication March 22, 2004 and
in revised form March 17, 2005.
 44
 
J. Aquat. Plant Manage.
 
 43: 2005.
 
RESULTS AND DISCUSSION
 
After incubating the plates for 7 d, inhibitory plaques
(zone plaques) of different sizes and shapes were observed
on top of the agar plate (Table 1). Control disks resulted in
no plaque formation. 
 
Rhus trichocarpa
 
, 
 
Quercus variabilis
 
 and
 
Mallotus japonicus
 
 produced larger diameter plaque zones
than the others (Table 1, Figure 2). Since the leaf disk sur-
face was initially sterilized, plaque formation is likely due to
its exudates and not of the action of microorganisms. The
formation of plaque zones suggests the presence of active al-
lelopathic compounds in the leaf litters of the tested species.
Additionally, the variation in sizes and shape of plaque
zones may indicate the activity-dependent nature of the leaf
litter species. Evaluation of the relationship in size between
leaf disks and plaque zones of 
 
R
 
.
 
 trichocarpa
 
 (most active) and
 
Ficus erecta
 
 Thunb. (least active) showed that both parame-
ters are highly correlated (r = 0.899) in the former, but not (r
= 0.252) in the latter (Figure 3), indicating that the rate of
diffusion depends on the specific leaf disk area and species.
 
R
 
.
 
 trichocarpa
 
, 
 
Q
 
. 
 
variabilis
 
 and 
 
M
 
. 
 
japonicus
 
 showed large,
clear plaque zones, suggesting the presence of strongly active
allelopathic substances effective against 
 
M. aeruginosa
 
.
Leaf litters may strongly influence algal communities es-
pecially in shallow lakes and reservoirs (Gross 2003). There
are numerous reports regarding allelopathic effects from
leaf litter leachates. For example, it has been known for
many years that poor plant growth under the black walnut
trees (
 
Juglans nigra
 
 L.) and the Japanese red pine trees (
 
Pi-
nus densiflora
 
 Sieb. et Zucc.) are due to allelopathic effect of
their leaf litters (Massey 1925, Lee and Monsi 1963). How-
ever, reports that discuss the relationship between leaf litter
leachates and aquatic organisms hardly exist. This study pro-
vides a potential method for improving investigations regard-
ing the influence of shoreline and littoral zone vegetation on
the occurrence of water blooms.
Results of this study suggest that deciduous leaf litters ac-
cumulating on littoral zones of lakes and reservoirs contain
active compounds which are inhibitory to the growth of
 
M. aeruginosa
 
, and that leaf litter from 
 
R
 
. 
 
trichocarpa
 
, 
 
Q
 
. 
 
vari-
abilis
 
 and 
 
M
 
. 
 
japonicus
 
 can be potential natural inputs in con-
trolling their excessive growth. More studies of this nature
Figure 1. Schematic diagram of the assessment method used in this study. Leaf disks sterilized with ultraviolet irradiation (257.3 nm, 6 h for each faces) were
placed on the top layer of cyanobacteria (M. aeruginosa). Paper disks sterilized with heat (150 C, 6 h) were used for control. Cell numbers per plate calcu-
lated by the growth curve are 4.78 × 107 (assumed 50% recovery rate). After incubation for 7 d, the plaque zone diameter was measured in mm.
 J. Aquat. Plant Manage.
 
 43: 2005. 45
are necessary to elucidate further the extent and nature of
the inhibition. Other cyanobacteria species should be tested
as well. The inhibitory effect of deciduous leaf litters based
on their decomposition rates should also be investigated.
For the suppression of water blooms, the long-term goal
remains that of reducing nutrient inputs, but, even if this is
achieved, continued management will often be necessary to
maintain lakes and reservoirs in the “desired” state. Natural
leaf litter input is one factor that may help to minimize the
likelihood of excessive algal growth. The bioassay system in-
troduced here could be a useful tool for providing informa-
tion that will be helpful in determining the role of shoreline
vegetation in impacting algal communities in littoral zones
of lakes and reservoirs.
 
ACKNOWLEDGMENTS
 
We are grateful to Microbial Culture Collection, National
Institute for Environmental Studies, Japan, for supplying
 
M
 
. 
 
aeruginosa
 
 and for giving us their technical advice.
 
LITERATURE CITED
 
Carmichael, W. W. 1992. A status report of planktonic cyanobacteria (blue-
green algae) and their toxins. United States Environmental Protection
Agency 600/R-92/079:32-33.
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Gross, E. M. 1999. Allelopathy in benthic and littoral areas: case studies on
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In:
 
 K. M. M. Inderjit Dakshini and C. L. Foy (eds.).
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T
 
ABLE
 
 1. L
 
IST
 
 
 
OF
 
 
 
SAMPLES
 
 (
 
LEAF
 
 
 
DISKS
 
) 
 
WITH
 
 
 
THEIR
 
 
 
CORRESPONDING
 
 
 
PLAQUE
ZONE
 
 
 
DIAMETER
 
 
 
AND
 
 
 
CHARACTERISTICS
 
. D: T
 
HE
 
 
 
PLAQUE
 
 
 
BOUNDARY
 
 
 
IS
 
 
 
DISTINCT
 
.
ND: T
 
HE
 
 
 
PLAQUE
 
 
 
BOUNDARY
 
 
 
IS
 
 
 
NOT
 
 
 
DISTINCT
 
. P: T
 
HE
 
 
 
PLAQUE
 
 
 
SHAPE
 
 
 
IS
 
 
 
PROTEAN
 
.
Sample
Plaque zone
Diameter (mm) Characteristics
 
Rhus trichocarpa
 
 Miq. 65 ND, P
 
Quercus variabilis
 
 Blume 64 D
 
Mallotus japonicus
 
 (Thunb.) Muell. Arg. 60 ND, P
 
Vaccinium oldhami
 
 Miq. 49 D
 
Rhus succedanea
 
 L. 46 D
 
Viburnum erosum
 
43 D
 
Prunus lannesiana
 
 (Carr.) Wils. 40 D
 
Vaccinium ciliatum
 
35 D
 
Castanea crenata
 
 Sieb. et Zucc. 34 D
 
Vaccinium smallii 
 
A. Glay var. 
 
glabrum
 
 Koidz. 29 D
 
Parabenzoin praecox
 
 (Sieb. et Zucc.) Nakai 29 D
 
Salix caenomeloides
 
 Kimura 28 D
 
Quercus acutissima
 
 Carruth. 28 D
 
Photinia glabra
 
 (Thunb.) Maxim. 28 ND
 
Lyonia neziki
 
 Nakai et Hara 27 D
 
Vitis coignetiae
 
 Pulliat 25 D
 
Rhus javanica
 
 L. 23 D
 
Ilex pedunculosa
 
 Mip. 20 D
 
Quercus glauca
 
 Thunb. 20 D
 
Quercus aliena
 
 Blume 14 D
 
Ficus erecta
 
 Thunb. 12 D
 
Wisteria brachybotrys
 
 Sieb. et Zucc. 0 Not measurable
Control 0 Not measurable
Figure 2. Growth response of cyanobacteria to leaf disks. Disks were placed
on the top layer of cyanobacteria (M. aeruginosa). After incubation for 7 d,
plaque zone formation around the disks was examined. Leaf disk: Rhus tri-
chocarpa. Control: Paper disk.
Figure 3. Relationship between the leaf disk (mm2) and the plaque zone size
(mm2). Rhus trichocarpa (): r = 0.899, n = 16, p < 0.001; Ficus erecta (): r =
0.252, n = 14. Error bars show standard errors.
 46
 
J. Aquat. Plant Manage.
 
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 forest
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 L. and 
 
J. cinera
 
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Assessment Method for Leaf Litters Allelopathic Effect on Cyanobacteria

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