Vol.2, No.4, 491-497 (2011)
opyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
Agricultural Scienc es
Evaluation of the toxicity of Streptomyces aburaviensis
(R9) extract towards various agricultural pests
Ismail Saadoun1*, Sereen Bataineh2, Qutaiba Ababneh2, Khalid Hameed2, Kevin Schrader3,
Charles Cantrell3, Franck Dayan3, David Wedge3
1Department of Applied Biology, College of Sciences, University of Sharjah, Sharjah, Sharjah-United Arab Emirates;
*Corresponding Author: isaadoun@sharjah.ac.ae
2Department of Applied Biological Sciences, Faculty of Science and Arts, Jordan University of Science and Technology, Irbid, Jordan;
3USDA, ARS, Natural Products Utilization Research Unit, Thad Cochran Research Center, University Avenue, Minneapolis, USA.
Received 7 September 2011; revised 19 October 2011; accepted 27 October 2011.
The dichloromethane extract of culture filtrate
from Streptomyces aburaviensis R9 was evaluated
using various rapid bioassays to determine po-
tential inhibitory effects towards phytopathogenic
fungi (Colletotrichum acutatum, C. fragariae, C.
gloeosoprioids, Botrytis cinerea, Fusarium oxy-
sporum, Phomopsis viticola and P. obscurans),
fish bacterial pathogens (Edwardsiella ictaluri
and Flavobacterium columnare), a green alga
(Selenastrum capricornutum), plant seeds [Bent
grass (Agrostis sp.) monocot and lettuce (Lactuca
sativa) dicot] and 2-methylisoborneol (MIB)-pro-
ducing cyanobacteria (Planktothrix perornata and
Pseudanabaena sp.). The dichloromethane extr-
act showed selective inhibition against the
cyanobacterium P. perornata, w ith a lowest-com-
plete-inhibition concentration (LCIC) of 10 mg/L
and lowest-observed-effect concentration (LOEC)
of 10 mg/L while LCIC and LOEC values were
100 mg/L when tested a g ains t S. capr icornutum.
This extract also showed slight meristematic
cytogenic necrosis at 200 mg/L towards ger-
minated seeds of bo th test plant s. The compou nds
were not very toxic towards the channel catfish
(Ictalurus punctatus) pathogenic bacteria E. ic-
taluri and F. columnare. Preliminary evaluation
of the extract toward C. acutatum, C. fragariae
and C. gloeosoprioids using TLC bioautography
revealed moderate activity. How ever, further eva-
luation of the extract using a microtiter plate
bioassay determined that inhibition was strongest
against C. acutatum and C. fragariae, though
this inhibitory activity diminished at 72 hours
and was moderately less active than the com-
mercial fungicides azoxystrobin and captan when
comparing 1 - 100 mg/L levels at 48 hours.
Keywords: Algae; Catfish; Cyanobacteria; Fungi;
Pathogens; Streptomyces
Biological methods and technologies in coordination
with agricultural production [1,2] were established to
solve problems of the deleterious effects of chemical
fertilizers and pesticides on the environment and the
appearance of organisms with resistance resulting from
the high application rates of currently used agrochemicals
[3-5]. Such chemicals are widely used despite the grow-
ing public concerns of several undesirable consequences
of using them such as accumulation in the environment,
biomagnification and excessive persistence [6].
Microorganisms are ubiquitous in nature and well-
known to produce bioactive materials of particular prac-
tical value. Among these beneficial microorganisms are
the actinomycetes, a group of filamentous bacteria which
include many species that are characterized by the pro-
duction of important extracellular bioactive compounds
including antibiotics [7,8]. The majority of those isolates
producing bioactive compounds belong to species within
the genus Streptomyces, and several Streptomyces spp.
have been advocated as promising biocontrol agents
against several phytopathogenic fungi and bacteria [9-14].
In addition, members of the genus Streptomyces are well
known for their potential to produce herbicides [5,15].
Natural compounds, such as the secondary metabolites
of actinomycetes and particularly streptomycetes, have
been demonstrated to be bioherbicides and include ani-
somycin, bialaphos, herbicidans A and B [5]. Therefore,
attempts have been made to isolate streptomycetes with
bioherbicidal properties.
Several studies have previously been conducted with
soil streptomycetes isolated in Jordan for their potential
to produce antibiotics [16-18]. In a recent study by
I. Saadoun et al. / Agricultural Sciences 2 (2011) 491-497
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
Bataineh et al. [19], the herbicidal activity of streptomy-
cetes isolated from different habitats in Jordan were
evaluated for their phytotoxic potential against common
broad leaf and grass. In addition, the optimal medium
suitable for higher phytotoxic activity in the culture fil-
trate of isolates and the method for extraction of poten-
tial phytotoxic compounds were investigated.
The present study was conducted to extend that inves-
tigation by determining the activity of the cell-free cul-
ture extract of one streptomycete isolate (Streptomyces
aburaviensis R9) against various agricultural pests. In
addition to potential herbicidal activity, the extract was
evaluated using rapid bioassays for activity against
off-flavor compound-producing species of cyanobacteria
(blue-green algae), bacterial pathogens of catfish, and
fungal phytopathogens in order to determine if other
useful bioactive compounds (toxins) were produced by S.
aburaviensis R9.
2.1. Extract Preparation
Extracts from S. aburaviensis R9 were obtained ac-
cording to Mallik [15]. Isolate R9 (7-day growth) from
an agar plate was used to inoculate a flask containing 25
mL of GPM broth (10 g/L glucose, 5.0 g/L peptone and
20.0 g/L molasses). This primary culture was incubated
at 28˚C with shaking at 110 rpm for 5 days inside an
orbital shaker incubator (TEQ, Portugal), after which the
primary culture was used to inoculate a 500-mL mass
culture of GPM broth. The mass culture was incubated
for 7 days under the same conditions used for the pri-
mary culture. Mass culture cell-free filtrate was ex-
tracted with 100% dichloromethane (1:3 v/v) (Acros
Organics, USA). The solvent was evaporated using a
rotary evaporator (Heidolph, Germany) at 29˚C. The
residues were then dissolved in 4 mL 100% dichloro-
methane and transferred into sterilized test tubes. To
determine the total solid content of the extract, the dis-
solved residue was dried in a jet stream of N2 gas.
2.2. Herbicide Bioassay
A standardized 24-well microtiter plate assay [20] was
used to determine the R9 extract activity towards mono-
cot (Agrostis stolonifera L. bentgrass) and dicot (Lac-
tuca sativa L. lettuce) representative plant types. The
wells in the microplate were prepared by adding 20 μL
of each of the test solutions of the extract to filter paper,
allowing the filter paper to dry and then adding 200 μL
of water. Final exposure concentrations were 0.2, 2.0,
20.0, and 200.0 mg/L.
2.3. Algaecide Bioassay
The methods outlined in Schrader et al. [21] were
used to evaluate the crude extract for selective toxicity
towards the 2-methylisoborneol (MIB)-producing-cyano-
bacteria [Planktothrix perornata [Skuja] Anagnostidis
and Komárek (syn. Oscillatoria perornata)], isolated
from a Mississippi, USA, catfish pond, and Pseudana-
baena sp. (strain LW397) [Skuja] Anagnostidis and Ko-
márek, isolated from a municipal drinking water res-
ervoir in Virginia, USA. The green alga (Selenastrum
capricornutum) [Printz] was included as a representative
of division Chlorophyta and to determine selective tox-
icity of the test extract.
The crude extract of S. aburaviensis R9 and the ex-
tract of the media (control) were dissolved separately in
100% dichloromethane (HPLC grade) to provide stock
solutions of 2.0, 20.0, 200.0, and 2000.0 mg/L. Final test
concentrations in microplate wells were 0.1, 1.0, 10.0,
and 100.0 mg/L. Absorbance measurements of microplate
wells were made every 24 h for 4 days using a Packard
model SpectraCount microplate photometer (Packard
Instrument Co., Downers Grove, Illinois, USA) at 650
nm. Three replications were used for each extract and
control concentration and experiments were repeated.
Mean absorbance measurements were calculated and
graphed to determine the lowest-observed-effect con-
centration (LOEC) and lowest-complete-inhibition con-
centration (LCIC).
2.4. Bactericide Bioassay
The same procedures used by Schrader and Harries
[22] were used to evaluate the crude extract of S.
aburaviensis R9 for antibacterial activity towards the
catfish pathogenic bacteria Edwardsiella ictaluri and
Flavobacterium columnare isolate 1016, except that a
96-well quartz microplate (Hellma Cells, Inc., Forest
Hills, New York, USA) was used to perform the bioassay
because the dichloromethane loading solvent is income-
patible with polystyrene microplates. Stock solutions
and final concentrations of the crude extract and media
extract (control) were the same as those used in the al-
gaecide bioassay.
2.5. Fungicide Bioassay
Isolates of Colletotrichum acutatum Simmonds, Col-
letotrichum fragariae Brooks, and Colletotrichum gloeo-
sporioides (Penz.) Penz. and Sacc. in Penz. were ob-
tained from Barabara J. Smith, USDA, ARS, Small Fruit
Research Station, Poplarville, Mississippi, USA. Colle-
totrichum fragariae (isolate CF63), C. acutatum (isolate
CAGoff), and C. gloeosporioides (isolate CG162) were
used for the pathogen and bioautography studies. Isolate
I. Saadoun et al. / Agricultural Sciences 2 (2011) 491-497
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
CF63 is one of the most virulent isolates that infects
strawberry plants and induces both crown and fruit rot
[23]. CF63, CAGoff, and CG162 were used as standard
test isolates because of our extensive knowledge of these
isolates and their known fungicide sensitivity profiles in
both bioautography and microtiter formats. The three
Colletotrichum species were isolated from strawberry
(Fragaria × ananassa Duchesne). Bo try tis cin erea Pers.:Fr,
was isolated from commercial grape (Vitis vinifera L.)
and Fusarium oxysporum Schlechtend:Fr from orchid
(Cynoches sp.) by D. E. Wedge, USDA ARS Natural
Products Utilization Research Unit, University, Missis-
sippi, USA. Phomopsis viticola (Sacc.) and P. obscurans
(Ellis and Everh) Sutton were obtained from Mike A.
Ellis, Ohio State University, Wooster, Ohio, USA. Fungi
were grown on potato-dextrose agar (PDA, Difco, De-
troit MI) in 9 cm petri dishes and incubated in a growth
chamber at 24˚C ± 2˚C and under cool-white fluorescent
lights (55 ± 5 µmols·m–2·sec–1 light) with 12 h photope-
Conidia were harvested from 7 - 10 day-old cultures
by flooding plates with 5 mL of sterile distilled water
and dislodging conidia by softly brushing the colonies
with an L-shaped glass rod. Conidial suspensions were
filtered through sterile miracloth (Calbiochem-Novabio-
chem Corp., La Jolla, California, USA) to remove myce-
lia. Conidia concentrations were determined photomet-
rically, from a standard curve based on the absorbance at
625 nm and suspensions were then adjusted with sterile
distilled water to a concentration of 1.0 × 106 co-
Bioautography procedures of Meeaza et al. [24] and
Tabanca et al. [25] for detection of naturally occurring
antifungal agents were used to evaluate antifungal activ-
ity of the dried dichloromethane cell-free culture extract
of R9 and GPM broth. Samples were prepared in di-
chloromethane at a concentration of 2000 mg/L and 4
and 8 µL were used in a dose-response to evaluate each
sample. Conidia of Colletotrichum fragariae, C. acu-
tatum and C. gloeosporioides suspensions were adjusted
to 3.0 × 105 conidia/mL with liquid potato-dextrose
broth (PDB, Difco, Detroit, Michigan, USA) and 0.1%
Tween-80. Using a sterile 50-mL chromatographic sp-
rayer, each glass silica-gel thin-layer chromatography
(TLC) plate with fluorescent indicator (250 mm, silica
gel GF Uniplate, Analtech, Inc., Newark, Delaware, USA)
was sprayed lightly (to dampness) three times with the
conidial suspension. Inoculated plates were placed in a
30 × 13 × 7.5 cm moisture chamber (100% RH, 398-C,
Pioneer Plastics, Inc., Dixon, Kentucky, USA) and in-
cubated in a growth chamber at 24˚C 1˚C and 12 h
photoperiod under 60 5 µmols·m–2·sec–1 light. Inhibi-
tion of fungal growth was measured 4 d after treatment.
Sensitivity of each fungal species to each test compound
was determined by comparing size of inhibitory zones.
Means and standard deviations of inhibitory zone size
were used to evaluate antifungal activity of essential oils,
solvent fractions, and pure compounds. Bioautography
experiments were performed multiple times using both
dose- and non-dose-response formats. Fungicide techni-
cal grade standards benomyl, cyprodinil, azoxystrobin,
and captan (Chem Service, Inc., West Chester, Pennsyl-
vania, USA) were used as controls at 2.0 mM in 2.0 µL
of ethanol.
A standardized 96-well microtiter plate assay devel-
oped for discovery of natural product fungicidal agents
[26,27] was used to determine sensitivity of B. cinerea,
C. acutatum, C. fragariae, C. gloeosporioides, F. ox-
ysporum, Phomopsis viticola, and P. obscurans to the
various antifungal agents in comparison with known
fungicidal standards. Captan and azoxystrobin were used
as standards in this experiment. Each fungus was chal-
lenged in a 6-point dose-response format using test com-
pounds where the final treatment concentrations were
0.01, 0.1, 1.0, 10.0 and 100.0 mg/L. Microtiter 96-well
quartz plates (Hellma Cells, Inc., Forest Hills, New York,
USA) were covered with a plastic lid and incubated in a
growth chamber as described previously for fungal
growth. Growth was then evaluated by measuring ab-
sorbance of each well at 620 nm using a microplate
photometer (Packard Spectra Count).
Chemical sensitivity of each fungus was evaluated
using 96-well plate microbioassay format. Each chemi-
cal was evaluated in duplicate at each dose (0.01, 0.1,
1.0, 10.0 and 100.0 mg/L). Mean absorbance values and
standard errors were used to evaluate fungal growth at
48 h and 72 h, except for P. obscurans and P. viticola the
data were recorded at 120 h. Analysis of variance of
means for percent inhibition of each fungus at each dose
of test compound (n = 4) relative to the untreated posi-
tive growth controls (n = 32) were used to evaluate fun-
gal growth inhibition. Each test fungicide was run in
duplicate at each concentration, and the experiment was
repeated three times.
3.1. Extract Yields from Cultures of
Streptomyces aburaviensis R9
Table 1 shows the yield of the concentrated dichloro-
methane cell-free extracted broth (mg) of S. aburavie nsis
R9 culture from different batches. An average of 138 mg
of dried cell-free dichloromethane crude extract was
obtained per 500 mL of GPM culture broth. However,
the media extract (control) yielded an average 120 mg of
dried extract per the same volume. This difference in the
I. Saadoun et al. / Agricultural Sciences 2 (2011) 491-497
Copyright © 2011 SciRes. http://www.scirp.org/journal/AS/Openly accessible at
dried weight yields is likely due to the growth and me-
tabolites (e.g., phytotoxin) production by S. aburaviensis
R9 when grown in GPM broth [19].
3.2. Herbicide Bioassay
In the study by Bataineh et al. [19], a total of 231 dif-
ferent soil Streptomyces isolates were assessed for their
phytotoxic activity on seeds of cucumber and ryegrass
on the basis of suppressed seed germination, discolora-
tion of the root tip, reduced root and shoot growth and
eventual death of the root. The phytotoxicity symptoms
observed in the study by Bataineh et al. [19] were repre-
sented by discoloration and death of the root tips, and
these symptoms were profoundly evident when the same
isolate used in the current study, S. aburaviensis R9, was
evaluated for activity. The results suggested that the
phytotoxic effect is cytotoxic and affects the meriste-
matic cells due to production of an extracellular agent(s)
or toxin(s) by the bacterium.
The results of the current investigation determined
that there was phytotoxic activity of the dichloromethane
culture filtrate crude extract at 200 mg/L towards the
germinated seeds of both test-plant types [A. stolonifera
(monocot) and L. sativa (dicot)] (Ta b l e 2 ). None of the
lower test concentrations of the S. aburaviensis R9 ex-
tract were phytotoxic. In accordance with the bioassay
protocol, test results were reported in tenfold dilutions of
the test material (e.g., extract). Overall, the results from
this bioassay indicate that the S. aburaviensis R9 extract
was weakly phytotoxic activity towards both the mono-
cot and dicot plants tested because only the highest test
concentration inhibited growth.
3.3. Algaecide Bioassay
The dichloromethane extract showed selective toxicity
against P. perornata with a lowest-complete-inhibition
concentration (LCIC) of 10 mg/L and lowest-observed-
effect concentration (LOEC) of 10 mg/L (Table 3). For S.
capricornutum, the LCIC and LOEC values for the R9
extract were 100 mg/L, an order of magnitude less toxic
which exemplifies the selective toxicity of the extract. It
is interesting to note that the Pseud anabaen a sp. was
inhibited by 10 mg/L of the GPM broth medium extract
(without S. aburaviensis-(R9) culture). Therefore, an
ingredient in this media appears to be toxic towards this
species of cyanobacteria. However, such effect against
the green alga S. capricornutum was only at 100 mg/L as
indicated by the LCIC and LOEC results. In accordance
with the bioassay protocol, test results were reported in
tenfold dilutions of the test material (e.g., extract).
Table 1. Yield of the concentrated dichloromethane cell-free extracted broth (mg) of S. aburaviensis R9 culture from different
Weight of concentrated dichloromethane cell free broth (mg)
Batch No. Test 1 Test 2 Test 3 Average weightb Controla
1 126 154 141 140 ± 14 110
2 187 114 107 136 ± 44 130
aThe control and tests volumes were 500 mL; bThe average ± standard deviation were calculated from the tests of the same batch.
Ta b l e 2 . Growth observation of monocot and dicot plants at different concentrations of the concentrated dichloromethane cell-free
extracted broth of S. aburaviensis R9 culture.
R9 culture filtrate extract GPM broth medium extract
(mg/L) Lactuca sativa L
(lettuce, dicot) Agrostis stolonifera L
(bentgrass, monoco t) Lactuca sativa L
(lettuce, dicot) Agrostis stolonifera L
(bentgrass, monoco t)
0.2 0a 0 0 0
2.0 0 0 0 0
20.0 0 0 0 0
200.0 1 1 0 0
a0: No effect, 1: Complete inhibition of growth; Growth was observed after 7 days.
Ta ble 3. Activity of S. aburaviensis R9 culture filtrate extract on cyanobacteria and green alga as compared to the broth medium
Planktothrix perornata Selenast rum capricornutum Pseudanabaena LW397
Compound mg/L mg/L mg/L mg/L mg/L mg/L
R9 culture filtrate extract 10 10 100 100 10 10
GPM broth medium extract >100 >100 >100 >100 10 10
aLOEC: lowest-observed-effect concentration; bLCIC: lowest-complete-inhibition concentration.
I. Saadoun et al. / Agricultural Sciences 2 (2011) 491-497
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3.4. Bactericide Bioassay
The extracts were not very toxic at the concentrations
tested towards the catfish pathogenic bacteria Edward-
siella ictaluri and Flavobacterium columnare isolate
1016 (data not shown). For this bioassay, only activity
below 100 mg/L for IC50 and MIC values is considered
3.5. Fungicide Bioassay
Results of the preliminary evaluation of the S. ab-
uraviensis R9 extract using TLC plates on Colletroti-
chum acutatum (CaGoff); C. fragariae (Cf63) and C.
gloeosporioides (CG162) showed strong activity against
these fungal isolates. However, results from the micro-
titer-plate bioassay found the most significant activity
was towards C. acutatum and C. fragariae (Table 4).
Generally, as the test concentration of culture extract
increased, the percentage of growth inhibition of C. acu-
tatum and C. fragariae increased. Also, the culture ex-
tract was moderately less active than the commercial
fungicides azoxystrobin and captan when comparing 1,
10 and 100 mg/L treatments at 48 h. There was less in-
hibition of C. acutatum and C. fragariae by the culture
extract at 1, 10, and 100 mg/L at 72 h compared to 48 h
results (Table 4). This loss of activity could be due to
hydrolyzation of the active compound by the fungal spe-
cies, thereby making it less active. This can occur with
many commercial fungicides which actually make them
more fungistatic than fungicidal at the concentrations
that they are applied. However, it is somewhat difficult
to infer a direct comparison since we are comparing a
crude extract to pure compounds. The variations of per-
cent growth inhibition observed for the different dilu-
tions of the control broth were attributed to incomplete
solubilization of the control broth extract that was en-
countered when conducting the fungicide bioassay. It is
interesting that the broth medium extract also inhibited
the cyanobacterium Pseudanabaena sp. used in this
Table 4. Evaluation of the crude extract of S. aburaviensis R9 culture fractions for fungicidal activity towards different fungal phy-
topathogens at different concentrations as compared to known fungicidal standards, azoxystrobin and captan.
% Inhibitiona,b
(mg/L) Sample C. acutatum C. fragariae
Culture extract 4.5 (0) 1.0 (1.3)
Control broth 10.0 (7.2) 12.4 (8.0)
Azoxystrobinc 5.3 (0) 0 (0)
Captanc 2.7 (0) 4.5 (7.3)
Culture extract 17.4 (18.4) 0 (0)
Control broth 0 (0) 22.3 (20.7)
Azoxystrobin 37.6 (6.7) 47.5 (36.8)
Captan 5.1 (0) 19.3 (14.8)
Culture extract 5.1 (0) 21.0 (15.4)
Control broth 5.9 (14.3) 0 (3.2)
Azoxystrobin 75.2 (26.9) 77.2 (58.8)
Captan 12.2 (2.2) 26.6 (21.8)
Culture extract 13.8 (0.06) 53.9 (30.5)
Control broth 0 (0) 25.3 (25.9)
Azoxystrobin 77.9 (39) 76.98 (63.5)
Captan 72.5 (43.9) 59.4 (9.5)
Culture extract 45.5 (0) 66.4 (14.0)
Control broth 0 (0) 21.3 (22.0)
Azoxystrobin 86.6 (64.7) 82.0 (68.2)
Captan 100.0 (95.6) 100.0 (100.0)
Culture extract 78.7 (27.4) 91.6 (20.8)
Control broth 12.3 (15.0) 17.0 (24.3)
Azoxystrobin 88.7 (78.0) 85.9 (73.5)
Captan 98.1 (98.0) 99.2 (99.8)
aSample results only indicate inhibition. Zero (0) does not indicate the degree of stimulation, only that there was no inhibition. bNumbers not enclosed in paren-
theses = 48 h results; numbers enclosed in parentheses = 72 h results. cInternal standard compound(s) utilized in the 96-well assay.
I. Saadoun et al. / Agricultural Sciences 2 (2011) 491-497
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The antifungal activity of the culture extract is likely
due to the production of an antibiotic, though such a
metabolite produced by S. aburaviensis R9 appears to
not be a broad-spectrum antibiotic, but more specific
towards certain species of fungi. While growth inhibition
of some of the test species of fungi (e.g., Colletrotichum
acutatum (CaGoff); C. fragariae (Cf63) and C. gloeo-
sporioides (CG162) occurred in the presence of the cul-
ture extract, there was little to no activity towards the
Gram-negative bacteria fish pathogens used in this study
(Edwardsiella ictaluri and Flavobacterium columnare
isolate 1016). A previous study by Raytapadar and Paul
[28] determined that another isolate of Streptomyces
aburaviensis from Indian soil produced an antifungal
antibiotic, and they identified the isolate as Streptomyces
aburaviensis var. ablastmyceticus (MTCC 2469). An-
other study by Thumar et al. [29] identified antibiotic
production by a halotolerant alkaliphilic Streptomyces
aburaviensis strain Kut-8 that inhibited the growth of the
Gram-positive bacterium Bacillus subtilis. At present, it
is unknown if the antibiotic production of S. aburavien-
sis R9 is similar or identical to those cited in the previ-
ous studies above. Future isolation and characterization
of the active antifungal metabolite(s) would determine
these properties. Additional studies would also aid in
determining if the active antifungal metabolite(s) pro-
duced by S. aburaviensis R9 is also responsible for the
phytotoxic activity observed towards the plants A. stolo-
nifera and L. sativa and the cyanobacterium P. perornata.
Mr. Dewayne Harries provided technical support for the fungicide
assay. Appreciation is extended to University of Sharjah/UAE and to
Jordan University of Science and Technology for administrative sup-
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