NeK
I'bytol.
( l y S S ) . 1 0 8 . 2 1 1 21 S
Early events of vesicular-arbuscular
mycorrhiza formation on Ri T-DNA
transformed roots
BY G . B E C A R D AND
J. A. EORTIN
Centre de Recherche en Biologie Forestiere, Faculte' de Foresterie et de Geodesie,
Universite' Laval, Ste-Foy, Quebec, GIK 1P4, Canada
{Received 26 June 1981; accepted 23 October 1987)
S V M M .\ R Y
.\n tn vitro system using Ri T-DN.A transformed roots and the vesiculat-arbuscular mycorrhizal fungus Gigaspora
margarita Becker & Hall has been developed to study the initial events of mycorrhiza formation. Sucrose, sodium
and phosphorus were found to be critical components of the n-iedium used to establish the dual culture. L'sing a
single spore as inoculum it vvas consistently possible to obtain colonization of a preselected point on the root and
to time the colonization process (within .S days), .'\hundant viable and aseptic spores can be obtained. The systet-i-i
IS especially appropriate for studying the triggering of the fungal biotrophy towards the root.
Key words: Transformed roots, Gigaspora mariiarita, in vitro endomycorrhizas, sporulation.
fungi. 1\) stud>- the timing of these events and to
define a way of identifying the factors involved in
Vesicular arbuscular mycorrhizas (VAM) are pre- the process, it is necessary to have the means to
valent on a wide range of vascular plants (Nicolson, reproduce consistently, under aseptic conditions, the
1967). They exert significant eflects on the physi- sequence of colonization steps taking place over the
ology of these plants (Abbott & Robson, 1984), and first hours and days of contact.
are associated with virtually all types of terrestrial
Mosse & Hepper (1975) reported the use of root
habitats except those where ectomycorrhizas and/or organ culture to obtain typical infections with
erieoid n-ivcorrhizas prevail (Mosse, Stribley & Le Glomus mosseae in axenic culture and Mugnier &
Tacon, 1981).
Mosse (1987) bave developed a method using Ri TThe axenic cultiv-ation of members of the family
DNA transformed roots that they have successfully
Endogonaceae forming VAM symbioses is an impor- used as a host for Glomus mosseae and Gigaspora
tant challenge from both the scientific and practical margarita. Our objecti\-e vvas to improve these
point ol view. However, axenic cultivation of these methods and to adapt them for the study of the initial
fungi, in total absence of a host, has yet to be events of VAM ontogenesis (0-6 days).
achieved. Empirical methods using diverse culture
media, including root extracts and root exudates,
have sot-i-ietimes shown a growth stimulation of the M . A T H R 1 . A I . . S . A N D M K T H O n S
fungi, but have never allowed the establishment of a Source of root organ culture
permanent pure culture (Hepper, 1984). Previous Transformed carrot {Datietis carota Iv.) roots were
observations have shown that an accelerated develop- prepared as follows: carrots were thoroughly
ment of the VAM mycelium takes place shortly after washed, peeled, soaked in 95 "o (v/v) ethanol for
the contact with the root (Mosse & Hepper, 1975);
10 s, surface sterilized in 1 "„ NaOCI for 15 min, and
this suggests tbat tbe critical exchange of chemicals rinsed in sterile distilled water before being sectioned
between symbionts takes place during the first few trans\-ersely into 5 n-it-n thick slices. Tbe slices were
hours or days after contact. We postulate that the in then in-imediately placed on 1 "„ water agar in Petri
sttii identification of the compounds exchanged dishes and inoculated with the A, Agrobacterium
between the host and the fungus is a prerequisite for rhizogenes strain (fron-i Dr L. Moore) on the distal
obtaining the in vitro axenic cultivation of VAM face of the sections (Ryder, Tate & Kerr, 1985). A
c-Ti O N
212
G. Recard and J. A. Eortin
Table 1. Composition of media*
MW
M
MMl
(mgl ')
(mgl ')
(mg 1-')
731
453
80
731
—
731
731
80
80
80
KCl
65
65
65
65
NaH.,PO,.2H.,O
21-5
MgSOj.7H2O
Na.,SO,.10H,,O
KNO.,
KH.,PO,
Ca(NO.,).,.4H.,O
Sucrose
NaFcEDTA
Kl
MnCl.,.4H2O
ZnSO,.7H.,O
H.HO,
CuS(|,.5H..()
Na.^Mo(), . i n / )
Clycine
Thiamine hydrochloride
Pyridoxine hydrochloride
N'icotinic acid
Myo inositol
Bacto .Agar
—
288
19-1
4-8
288
30000
8
0-75
288
10000
8
0-75
10000
8
0-75
6
2-65
MM2
(mg 1 ')
MM3
(mg 1 •)
731
453
80
65
4-8
288
30 000
8
0-75
4-8
288
10000
8
0-75
6
6
2-65
2-65
1-5
1-5
1-5
1-5
1-5
0-13
0-0024
3
0-13
0-0024
3
0-13
0-0024
3
0-13
00024
3
0-13
0-0024
3
0-1
0-1
0-1
0-5
50
10000
0-1
0-1
0-1
0-5
50
10000
0-5
50
10000
6
6
2-65
2-65
0-1
0-1
0-5
50
10000
0-1
0-1
0-5
50
10000
The pll of the media was adjusted to 5-5 before sterilization at 121 °C for 15 min.
* MW, modified Wbitc's mcdiut-n ; M, minimal medium; MMl, 2, 3, modified minimal media.
loopful ol bacterial suspension taken from a 2-dayold culture grown on Difco Nutrient Agar was used
as inoculum. Three weeks later, a few transformed
roots proliferating on the inoculated sections were
aseptically excised and transferred into Petri dishes
containing modified White's medium (MW, Table
1) supplemented with 500 mg F ' of carbenicillin.
Three successive subcultures were necessary to free
the transformed roots of bacteria. One root apex
from the final subculture was excised and grown on
fresh MW medium to initiate a clonal culture.
Confirmation of the transformation of the carrot
roots.
The
DNA
(T-DNA)
transferred
from
the Ri
plasmid oiAgrobacterium rhizogenes during transformation of the root induces the production of opines
(Tepfer & Tempe, 1981). To demonstrate the
production of opine, crude extracts (10-30 /i]) obtained from transformed roots and control non-transformed roots were spotted on chromatography paper
(Whatman 3 MM) and electrophoresed for 2 h at
400 V in presence of an acid buffer (1 M acetic acid,
titrated to pH 1-8 with formic acid). The electrophoretogram was stained with silver nitrate according to the technique of Trevelyan, Procter &
Harrison (1950). The identity of spots was established by comparison with appropriate standards such
as mannopine and agropine [provided by Dr P. Dion
who received mannopine from Dr W. S. Chilton;
partially purified agropine was synthesized from
mannopine according to the method of Petit et al.
(1983)]. Control tissues were obtained from rootlets
of germinated carrot seeds.
Culture media
Routine maintenance of transformed roots was made
on modified White's medium solidified with 1 %
Difco Bacto-agar (MW, Table 1). Since this medium
was demonstrated during the experiment to be
detrimental for the establishment of primary mycorrhizal infection in dual culture, a 'minimal' medium
(M) was defined for the study of primary mycorrhizal
colonization (Table 1). The ontogenesis of myeorrhiza formation was also compared using three
'modified minimal' media M M l , MM2 and MM3
(Table 1). The pH of all media was adjusted to 5 5
before sterilization at 121 °C for 15 min.
Root growth in the minimal medium (Af)
Elongation rate of roots was studied by placing seven
10 mm tips of lateral roots on M medium for 20 days
in inverted Petri plates. The linear elongation (mm)
of each individual root was measured every 2 days
and used to calculate the growth rate of the
corresponding tissue (mm d~') and to establish a
pattern of growth rates during the culture.
The fungal inoculum
Spores of Gigaspora margarita Becker & Hall
(DAOM 194757, deposited at the Biosystematic
Research Center, Ottawa, Canada) were recovered
VA mycorrhiza and trattsforttied roots
from a leek {Attiuttt porrtittt L.) pot culture by wet
sieving (Gerdeman & Nicolson, 1963) followed by
a density gradient centrifugation to furtber purify
tbe spores (Furlan, Bartshi & Fortin, 1980). Spores
were surface sterilized using a modification of the
tw-o-step procedure of Mertz, Heithaus & Bush
(1979). The steps were carried out aseptically in a
laminar flovy cabinet, except for tbe centrifugations.
Step 1. The spores were washed in a 0-05 °,, (v-/v-)
Tween 20 solution, soaked in a vacutainer tube
(Becton Dickinson) witb a 2'\, (w/v) cbloramine T
solution for 10 min, and rinsed three times by
centrifugation for 30 s in sterile distilled water. A
second treatment with chloramine T followed by
rinsing in water was performed in the same manner.
At this stage, spores could be stored at 4°C up to 5
months in sterile solution containing 200 mg 1 '
streptomycin and 100 m g T ' gentamycin.
Step 2. Stored spores were redisinfected bystirring in 2 ",, chloramine T and rinsing v\'ith water,
in the upper part of a sterile 022 //m filter holder
apparatus. Vacuum (40 cmHg) was applied in order
to remove the liquid phases. The final spore
suspension was concentrated by aspiration, then
30-60 spores per Petri plate were spread out on 1 "„
vyater agar. Following this second decontamination,
spores could be used immediately or after a few
weeks of storage at 4°C. Only wbite to creamcoloured spores were selected and picked up with a
fine sterilized paint-brush.
Estabtishtnent of a pritnary mycorrhizal cototiizatiott
Primary mycorrhizal colonization was achieved by
placing a single non-germinated spore with a single
transformed root in the same dish. Root explants
were initiated by excising 10 mm tips of lateral roots
taken from the clonal culture. Root tips were grown
either on M or MM media in inverted Petri plates at
25°C for 20 d, and occasionally on MM medium for
lOd.
A single spore of Gigaspora tnargarita was inserted
into the agar (prepared with either M or MM media)
at the bottom of a Petri plate, so that the germ tube
could grow toward the surface of the medium (about
4 days). Tbe exact location of emergence of the germ
tube at the surface of the agar was determined with
a binocular lens. Tbe root was then located in such
a way that the region where primordia for the lateral
roots are usually formed (4-5 and 35 cm behind the
tip, respectively for the 20-day-old and the 10-dayold roots) was over the emerging germ tube (Fig. 1).
The two partners were then treated as a single
experimental unit.
The ability of G. tnargarita to colonize tbe host
was compared on 10- and 20-day-old roots, but 20day-old roots were used exclusively vvben comparing
the effects of different media (M, M M l , MM2,
MM3) on mycorrhiza formation. Sampled root
segments were examined for mycorrbizal coloniza-
213
Figure 1. System used for establishment of primary
colonization. The germ tube growth of the Gigaspora
tnargarita spore is negatively geotropic and contacts the
post-elongation zone of the root. This system is treated as
an experimental unit.
tion by clearing them for 2 min in 10 "„ KOH (w/v),
rinsing tbem in water and staining tbem in 0-1 °o
chlorazol black E (w/v) for 2 h (Brundrett, Piche &
Peterson, 1984). In all experiments, root segments
vyere sampled six days after the initial contact, except
for the sequential observations on ontogenesis wbere
roots were sampled every day over a 6-day period.
The treatments were applied on 8-15 experimental
units and repeated at least twice. Three sequential
stages in the development of the infection were
distinguished after root staining:
(i) attachment of the fungus to the root surface
without any penetration of the root or inter
and intracellular spread (Fig. 4fl);
(ii) intercellular spread of hyphae following
attachment (Fig. 46);
(iii) intracellular spread with arbuscule formation,
following attachment and intercellular spread
(Fig. 4 ^ .
These stages were observed at the same magnification and in the same field of view but at tbree
different focal planes, and were considered as three
distinct variables.
Lotig-tertn devetoptnent of duat ciitture
All dual cultures were performed on M medium.
Four to six primary mycorrhizal colonizations per
Petri plate were initiated and the cultures were
maintained up to 7 months at 25 °C. The spores
were placed on the agar surface and the Petri plates
were vertically incubated directing the germ tubes
upwards towards perpendicularly laid down roots.
The development of extramatrical phases and sporulation were monitored using binocular or inverted
microscopes.
R I-: S II L T S
Root tratisfortnatioti
Tbe crude extracts from transformed roots contained
opines characteristic of Ri T-DNA transformed
tissues (Fig. 2). Both mannopine and agropine were
214
G. Becard and J. A. Eor tin
30 III
t
1 t
15
17
I TRANSFORMED
10 -
ROOTS
t
t
10 wl
\
\
5 \
CONTROL
30
0 1
3
5
7
AGROPINE
9
n
13
19
Days
Figure 3. Root elongation rates during 20 days of culture.
Each point is a mean value of 7 root elongation rates
(mm d ') measured every 2 days on minimal medium (M).
Vertical lines indicate san-iple standard deviations.
MANNOPINE
Figure 2. Electrophoretogram of crude extracts of normal
carrot roots (control) and of carrot roots transformed by
Agrohacterium rhizogenes. Standard agropine was partially
purified.
The elongation zone of the 2()-day-old roots had a
larger diameter than the 10-day-old roots.
Decontamination and germination of the spores
clearly detected. Fxtracts from control roots sbowed
no positive reaction.
The method used for surface sterilization was
efficient. Contaminated spores (less than 5",,) were
immediately discarded. A germination rate of up to
95 "/(, was ohtained irrespective of the medium tested.
The germ tube emerged in any direction, and after it
had grown to two or three times the diameter of the
spore, it became negatively geotropic as observed by
Watrud, Hcithaus & Jaworski (1978).
Root growth
Growth of the root organ cultures on MW mediun-i
was prolific and the roots appeared normal. On M
medium, root growth was not very abundant but still
significant and the root diameter was smaller. The
same morphological changes were observed on roots
growing on MMl and MM3 but not on MM2
medium, indicating a clear relationship with sucrose
concentration. After 20 days of culture on M
medium, individual roots were very similar, showing
a mean length of 190 mm with a low coefficient of
variation of 2-4"/,,. The rate of root elongation
(mm d ') followed a distinct pattern over the first 20
days of culture. Two different rates of root growth
were observed : 9 mm d' ' for root tissues formed
between days 5 and 13, and 12mmd~' for root
tissues formed after day 15 (Fig. 3). We postulate
that the first 15 days were an adjustment period
during which the excised 10 mm lateral roots became
main roots which produced many new lateral roots.
Effects of different media on the early stages of
eolonization
On the M medium, 83 "/„ of the roots became
colonized (Table 2). All hyphal attachments to the
root surface led to successful endomycorrhiza formation. All the modifications of the minimal medium
( M M l , MM2, MM3) had obvious negative effects
on the final percentage of colonization. Increases of
KH2^0,1 or of sucrose concentration in the minimal
medium (MMl and MM2) dramatically decreased
percent colonization to 0 and 7",, respectively, the
main effect being via prevention of byphal attachment. Presence of Na._,SO,, in the minimal n-iedium
Table 2. Effects of different media on different stages of colonization
Number of
experimental
units
M
MMl
MM2
MM3
12
14
15
10
Attachment
with no further
spread
Intercellular
spread
(%)
0
0
13
10
(%)
Intracellular
spread
("«)
Total colonization
(inter-f intracellular)
("«)
17
0
0
20
67
0
7
20
83
0
7
40
VA mycorrhiza and transformed roots
215
Table 3. Effects of root age on different stages of colonization
Root age
(d)
Number of
experimental
units
20
10
12
T a b l e 4. Colonization
Days after
contact
1
2
Attachment
with no furtber
spread
Intercellular
sptead
Intracellular
spread
Total colonization
(inter -|- intracellular)
12-5
62-5
17
75
process during the first 6 days after contact between the gertn tube and the root
Number of
experimental
units
Attachment
with no futther
spread
(%)
Ititercellular
spread
(%)
11
0
0
3
4
5
12
12
11
11
0
25
18
9
6
12
0
33
36
9
0
Table 5. Linear spread of the intracellular
colonization in a root, 5 and 6 days after the initial
contact between the germ tube and the root surface
Days after
contact
25
Number of
experimental
units
Average length
of tbe colonized
root tissue {/im)
867
1386*
Indicates significant difference (P = 00171, t test).
(MM3) decreased the percent colonization to 4 0 %
of the root, and negatively affected all steps of the
colonization process.
Effect of root age
The 20-day-old roots were much more efficient as
hosts for the fungus, with a colonization rate of 75 %
compared with 25 % for the 10-day-old roots (Table
3). For the latter, the main restricting step was the
attachment to the root surface.
Process of colonization
After the initial contact between the germ tube and
the root, the first hyphal attachment and/or intercellular colonizatioti took place on the third day.
This attachment came from a ramification of the
germ tube which continued its growth in a negative
geotropic direction after bypassing the root. Attachment was followed by intracellular colonization and
formation of arbuscules 2 days later. The data show
that about 50 % of the germ tubes attached them-
Intracellular
spread
("0/ ^
0
0
0
0
64
67
17
Total colonizatioti
(inter -(- intracellular)
(%)
0
0
0
0
73
83
selves to the root surface or began to colonize the
epidermis and cortex during day 3, and formed
arbuscules during day 5 (Table 4). About 20% of the
germ tubes attached to and penetrated the root
during day 5 while 10 % of the germ tubes were able
to induce intracellular colonization directly during
day 5. In the latter case, the attachment to the root
surface was limited to one appressorium without
hyphal proliferation. In general, the germ tube
produced branches on the root surface (day 3) with
many appressoria (Fig. 4a). No significant progress
was observed during days 4 and 6 of the colonization
process, except for more widespread intracellular
colonization on day 6 (Table 5).
Extramatrical phase and sporulation in dual culture
After primary mycorrhizal colonization had taken
place, rapid development of extramatrical hyphae
was observed. Clusters of thinner branched hyphae,
more or less septate, appeared sporadically but were
not seen prior to a primary infection (Fig. 4rf).
Secondary infections were rapidly established elsewhere on the root and the fungus spread throughout
the Petri plate.
Sporogenesis was regularly observed between the
first and the seventh month of dual culture (Fig. 4e).
These spores, twenty times the number of spores
used as inoculum, were demonstrated to be a reliable
source of aseptic fungal inoculum.
DISCUSSION
Establishment of mycorrhizas depended greatly
upon presence/absence and concentration of
NajSO,,, phosphorus, and sucrose in the culture
21 6
G. Beeard and J. A. Eortin
Figure 4. Development of intramatrical and extramatrical phase of Gigaspora ynargarita. {a) Hyphal
attachment to the root surface (first focal plane). Bar, 80/«m. {b) Intercellular spread of the fungus in the root
(second focal plane). Bar, 80/<m. (c) Intracellular spread of the fungus in the root (third focal plane). Bar,
60 jim. ((•/) Post-infection structure of more or less septate branched hyphae. Bar, 300 //m. {e) .Spore production
during seven months of dual culture. Bar, 5 mm.
medium. These factors acted on the root and/or
the fungal physiology. Sodium sulphate was detrimental to VAM establishment. This result agrees with
observations of Mosse & Phillips (1971) who observed that internal development of Endogone mosseae in
the root of Trifolium parznjiorum decreased when
sodium was present in the medium. Phosphorus is
the most widely studied element in mycorrhizal
research. High P levels in the soil have been shown
to decrease or eliminate mycorrhizal infection (Baylis, 1967). Under our experimental conditions, a
decrease in the concentration of P from 434 mg V" to
1-08 mg 1 ' was a determining factor in the achievement of successful colonization. The concomitant
reduction (6%) of potassium was considered negligible with regard to the total amount in the medium.
In addition, successful mycorrhizal colonization was
achieved by reducing the sucrose concentration fron-i
3 to 1 '/',, in the medium, although root growth and
diameter were also reduced. Anatomical changes in
excised tomato root, in relation to the sucrose
concentration in the culture medium, were also
observed by Street & McGregor (1952). Histological
or cytological studies of the root at different sucrose
concentrations might provide information on mycorrhizal receptivity.
Root physiology, i.e. root age, affected the mycorrhizal colonization. At the same target point on the
VA mycorrhiza and transformed roots
two types of roots (i.e. root tissue differentiated 3—Idays earlier), 20-day-old roots were more receptiv-e
to colonization than the 10-day-old roots. However
the more receptive tissue at the moment of mycorrhizal formation had grown faster (124 mm d ' )
than the less receptiv-e (8-6 mm d"'). 'Phe diameter of
the more rapidly growing tissue was also larger.
Further studies on anatomical or biochemical differences between these root of different ages could
provide a better understanding of root receptivity to
mycorrhizal colonization.
The evaluation of these ditferent factors on
mvcorrhiza formation, using very few fungal spores
and root-tip segments, was made possible because of
the following:
(i) Dev-elopment of a common medium for the
dual culture was not constrained by the nutritional
requirements of either organism. Ri T-DNA transformed roots have a great growth potential because
they are a tumoural tissue (Nester et al., 1984) and
consec]uently can tolerate changes in mediun-i composition. Also, germination of Gigaspora margarita
spores depends little on media composition (Siqueira, Hubbell & Schenk, 1982), and under our conditions, was independent of the type of medium.
(ii) The ren-iarkable consistency in the rate of root
tip growth, a phenotypic consequence of clonal
culture, and the ease of manipulation of germ tube
gr(3wth ol ry. margarita spores towards a selected
region of the root, allowed for standardization of
events leading to mvcorrhizal initiation. We consider
this latter characteristic valuable for future studies
on cellular or molecular interactions between the two
partners using biochemical or n-iicroscopic techniques.
Therefore, it was possible to find an appropriate
medium (M) on which 80",, of the root was infected
within 5 days and to have a reproducible patteri-i of
colonization. An interesting feature observed in the
colonization process was the occurrence of a 2 day
interval between the two consecutiv-e colonization
steps, contact-attachment and attachment-intracellular spread. This interval may be an adaptation period
for the dev-elopmcnt of recognition mechanisms or
the synthesis of enzymes. Under our experimental
conditions, fungal attachment to the root surface was
the step most sensitive to unfavourable conditions.
We therefore consider the initial 2-day period after
contact as the critical step in the interaction between
the two partners. No apparent interaction was
observed prior to contact. The germ tube elongation
of G. margarita spores was always negatively geotropic and never deviated in the presence of a root. This
is different from the observations of Koske (1982)
who observ-ed attraction of the germ tube to the
root, which was attributed to the action of volatile
products of the root.
A feature of the extramatrical phase of G.
margarita is the formation of branched and septate
217
hv-phae referred to in the literature as ' pre-infection
fan-like structures' or ' arbuscule-like structures'
(Powell, 1976; Mosse & Hepper, 1975). 'Fhese
structures are believ-ed to form when hyphae from
spores grow very close to the root and they have been
considered to be the site of cytologicai changes
necessary before hyphae fron-i spores become phvsiologically infective (Powell, 1976). In contrast, in our
study such structtires were observ-ed only after
colonization was established (5 days or more after
contact) and sometimes many millimetres away fron-i
the root. They are not to be confused with branched
hyphae attached to the root surface before infection
(Fig. 4a). In any case, branching of hyphae seems to
be a specific response to a more or less intimate
interaction vyith a root, such as arbuscuies in the root
cells, branched hyphae on the root surface, or the
mentioned fan-like structures.
In vitro sporulation of C margarita was previously
observed by Miller-Wideman & Watrud (1984).
Production of new spores was limited to an average
of 3-5 spores per plate and many were considered
abortive. In our study, production of spores depended on the type of medium used. The best medium
for colonization (M) was also found to be the best
medium for spore production (unpublished results).
More than 100 spores per plate could be obtained.
Although active fungal growth was a prerequisite,
sporulation occurred in older cultures when fungal
growth had slowed. In most cases, spores appeared
at the end of the carrier hypha just before a nonviable septate tip. The production of a large number
of new, non-abortive spores offers the possibility- for
future physiological and anatomical studies on the
biogenesis of spores, and perhaps the potential for
aseptic, large-scale production of inoculum.
A prerequisite and essential step for the above
discussed development of the fungal extramatrical
phase is hyphal growth. 'Phe initial signal(s) which
switches the germinative spore from dependence on
its reserves for growth to dependence on the host for
further fungal growth and sporulation remains to be
found. It this were known, it might be possible to
achieve pure culture of the fungus without the host,
l l i e simple system for monitoring the first events of
mycorrhizal establishment that we have developed
and described is a model for such studies.
A C K N O VV L E n G H M E N T S
The authors wish to thank Dr Suha Jabaji-Hare, Dr Keith
Kgger and Dr Sally Smith for reviewing and correcting the
manuscript. This research was supported by the Natural
Sciences and Engineering Research Council of Canada
(NSKRC grant A-3235 to J. Andre Fortin).
R E E V. R V. N C E S
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m y c o r r h i z a e on plant g r o w t h . I n ; VA Mycorrhiza
(Ed. by C.
21 8
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