421
Agriculture, Ecosysteas a n d Environment, 29 (1989) 421-427 E l s e v i e r S c i e n c e P u b l i s h e r s B.Vo, ~ t e r d a m Printed i n C z e c h o s l o v a k i a
PROPERTIES
OF
BACTERIA
THE MYPHOSPHERE
IN
OF
A
VESICULAR-
ARBUSCUIJLRNYCORtV~IlZAL FUNGUS V. Vangura 1, M.O. Orozco 2, O. Grauov& 1 and Z. P ~ i k r y l 1 IDepartment of Microbial Ecology. Institute of Microbiology, C z e c h o s l o v a k Academy o f S c i e n c e s 142 20 Prague 4, Vldenska 1083, C z e c h o s l o v a k i a 2Department o f S o i l E c o l o g y , I n s t i t u t e of Ecology and S y s t e m a t i c , Cuban Academy o f S c i e n c e s 10800 H a b a n a 8 , Cuba ABSTRACT
Three c o l l e c t i o n s of b a c t e r i a were i s o l a t e d from the hyphosphere of extramatrlcal mycelium of the vesicular-arbuscular mycorrhlzal (VAM) f u n g u s Gl0mps f a s c t c u l a t u m , growing i n slnubios~s w i t h r o o t s of Trtfolium renens, Hibiscus e l a t u s and Axonopus. q r a c i l . ! s . The presence of bacteria on the surface of hyphae was verified by scanning electron microscopy. All of the isolated bacteria were gram-negative rods. but no fluorescent pseudomonads were p r e s e n t . Most o3 them r e q u i r e d amino a c i d s ( t y r o s t n e , a s p a r t i c acid and ornithlne) and growth factor's. Our results show that VAN fungi select hyphosphere bacteria from rhizosphere bacteria previously selected by the plant. INTRODUCTION In a previous paper it was shown that on the
hyphal surfaces
of extramatrical
fungi (Orozco et al. 1984).
microorganisms ectomycorrhtzas stimulated
Garbaye
from inside and
found
m¥corrhlzal
the that
infection
and
m~ntel some of
bacteria were present mycelia of various V ~
Bowen
(1987) i s o l a t e d
of Rhtzopogon luteolus bacteria Ptnus
significantly
radiata.
On
the
contrary, Hetrick et al. (1987) observed s u p p r e s s i o n o f VRM fungi by a community o f s o i l microorganisms. O l i v e l r a e t a l . (1987) found increased growth and phosphate uptake of cassava when it was inoculated simultaneously with Acaulospora longula and Pseudomonas p u t i d a ° such effect
a known r h i z o p l a n e
was o b s e r v e d
with highly
and E n t r o p h o s p o F ~ c o l o n ~ l a n a . In t h i s s t u d y the properties, requirement determined
of
bacteria
l~olated
and co]pared with
rhizosphere, bacterta.
bacterium.
H o w e v e r . no
efficient
Glomus m a n i h o t i s
especially
the nutritional
from the surface
known p r o p e r t i e s
of hyphae were
of rhizoplane
and
422
HATERIALS AND HETHODS Soils from which
bacteria
Table 1. Glomus f a s c i c u l a t u m synthesis
with
plants
scanning e l e c t r o n et e l .
were isolated
Texter
throughout
microscopy
were
sensu thls
ere
Gerd. study.
described
described
was
in
used f o r
Procedures f o r
previously
(Orozco
1984).
Table 1. S o i l s end p l a n t s were isolated
from which r h i z o s p h e r e b a c t e r i a
Collections of i s o l a t e s
Soils
H
red lateritic s o t 1. (Cuba)
Plants
Hibiscus e l a t u s Sw.
pH - S . 0
B
ys I low s a n d y s o i l (Cube) pH -
A
Axonopus gracilis
S.9
brown sol1 (Czechoslovakia) pH • 6 . 8
Trtfolium p r e t e n s e L.
pH was measured i n water e x t r a c t Extra.trice1 water and a 0.5
m ~ c e l l a were washed from s o i l - 1.0 mg sample was c o l l e c t e d
with
(Taylor,
Petrl dishes
17 cm
in diameter.
w a t e r and
Agar medium T3
1951) was added t o t h e s u s p e n s i o n , t h o r o u g h l y mixed, and
the b a c t e r i a bacteria
were i n c u b a t e d a t 27 oc f o r 5 d a y s .
were
nutritional media o f
to
tap
under a d i s s e c t i n g
m i c r o s c o p e . The sample was shaken w i t h 10 ml o f s t e r i l e transferred
sterile
isolated
and
ulntalned
requirements of bacteria Taylor 1
was ~ a s u r e d
were
with various nutritional
by optical
density.
The c o l o n i e s o f
on the same medium. The determined
i n !~lqutd
additions
and ~ r o w t h
.
42
RESULT3 AND DISCUSSION A scanning
electron
VAM h y ~ h a e a n d (Fig.
1).
root
Bacteria
are
also
~o~tes
on
reported
al.
1974,
present
on
the to
van the
fungi
el~tus
root
with
surface
is
shown
cover
1 to 8 ~ of the
Vuurde surface
(Bednarova
et
et
al.
of
hyphae
al.
1979). of
1979).
A root of Hibiscus elatus with hyphae of Gloaus fasci~ulatum. Bacteria-like b o d i e s a r e p r e s e n t on h y p h a l surface. Bar i n d i c a t e s I ~. the
symbiosis
surface
with
strains
isolated
of
different
chosen bacteria
the
et
of Hibiscus
been
and phytopathogenic
From
30
have
(Bovira
saprophytic
~ig.1.
bacterla~like
Bacteria
surface
micrograph
were
and
bacteria
G.
flsciculatum
plants,
isolated.
properties
were gram-negative
proportion
of
addition
substrates
of
each
strains, is given
which
to
increase
in Table
strain
but
for were the
growing
collections
Each collection of
fluorescent pseudomonads. We t e s t e d the isolates
basic medium
three
hyphae
of randomly
was comprised were examined.
none of them belonged
growth
requirements.
stimulated nutritional
in
in
to The
growth
value
of All
by
of the
2. The effect of casamlno acids
was enhanced by the addition of yeast extract and/oF trypton.
3
424
Table 2. N u t r i t i o n r e q u i r e m e n t s o f b a c t e r i a I s o l a t e d t h e hyphosphere o f a m y c o r r h i z a l fungus Growth stimulation
from
(~)
Collection of isolates C
C Y T
58.6
B
53.3
-
73.3
A
30.7
-
65.4
vitamin
on g r o w t h
of
in their
g r o w t h by the c o m b i n a t i o n on t h e bacteria
amino acid
was t y r o s i n e ,
aspartlc
acid
growth
citrulline
strongly
of collection
bacteria growing
on
we
both
endophyte
hyphosphere bacteria
of
between
plants,
that
and
bacteri~
of
amino acids, Bacteria
B while
from
only
55
htstidlne
of collections
and B and
H.
three
conclude
of s i n g l e strains
stimulated
isolates
N
of
most e f f e c t i v e
hyphosphere
glutamine
the
effect
The
is
stimulation
two other
collection
dlfferences
endophyte,
the
stimulated
bacteria
individual
4.
into
(especially
for
The
of
Table
contrary,
stimulated
Erom observed
acids.
which along with
bacteria
A, bu~ R o t
collections
requirement
in
divided
hyphosphere
stimulation
is given
On t h e
of the h~)hosphere
of
o f amino
and ornithlne
collections.
amino acids,
in different
and B) d i f f e r e d
hyphosphere
twenty
stimulation
shown i n Table 3. B a c t e r i a
amino acids
-
free
of addition
groups,
versus A
soil
65.5
C ÷ Y ÷ T
48.2
The e f f e c t
all
C + T
H
not determined = Casamtno acids = Yeast extract = Trypton
three
C ÷ Y
the
the
but
with
selection
plant,
originate
the three
only
of bacteria
k~e
suggest
from r h l z o p l a n e
and t h a t d e f i n i t i v e
collections
selection
of
one f u n g a l depends
that
on
the
and r h i z o s p h e r e
o c c u r s on the h y p h a l
surface.
V~gnerov~ e t
al,
b a c t e r i a and one t h i r d while
Lochhead
(1940)
(1960)
found t h a t two t h i r d s
of r h l z o p l a n e b a c t e r i a found
of f r e e
soil
were g r a m - p o s i t i v e ,
that a substantial
portion
of the
425
Table
3o Amino a c i d r e q u i r e m e n t of bacteria isolated t h e h y p h o s p h e r e o~ a m y c o r r h i z a l gungus
Growth s t i m u l a t i o n
from
(~)
C611ectlon of isolates
~;
A2
A3
cl
H
30.4
4.3
4.3
26.1
8
84.2
78.9
68°4
5.2
A
30,4
60.8
26.0
8.6
A1 a t h r e o n i n e , o r n i t h i n e , g l ¥ c l n e , t y r o s i n e , c y s t l n e , a s p a r t l c acid. -alanine A2 ~ l y s i n e , methlonine, phenylalanlne, leucine, glutamine, histidine, citrullt~e A3 = v a l l n e , a r g i n t n e , i s o l e u c t n e , g l u t a m i c a c i d , s e r i n e , proline, aspartlc acid C1 - r e p r e s e n t s a group which was s t i m u l a t e d by casamlno a c i d s b u t n o t b y a n y o f t h e a m i n o a c i d s i n g r o u p s A 1 - A3 Table
4." P r e d o m i n a n t a m i n o a c i d r e q u i r e m e n t of bacteria from the hyphosphere of a mycorrhizal fungus
Growth stlmulatxon
(%)
Amino a c i d s H
B
A
41.6
69.2
30.7
Cltrulline
0
22.2
38.4
Glutamine
0
55.5
0
Histtdlne
0
23.0
53.8
Ornithtne
8.3
38.4
23.0
Tyrostne
75.0
76.9
23.0
Asparttc acid i
isolated
A26
r h t z o p l a n e com m uni t y was g r a m - n e g a t i v e bacteria. Our o b s e r v a t i o n that h y p h o s p h e r e bacteria were all gram-negative strongly supports the hypothesis
of step-wise
(free
soil
-) rhizosphere
-> h y p h o s p h e r e ) s e l e c t i o n o f b a c t e r i a . The f i r s t step - selection by plant - c o u l d o c c u r v i a r o o t e x u d a t e s ( e s p e c i a l l y amino a c i d s such as g l u t a m t n e shown b y Van~ura ( 1 9 8 8 ) . The d i v e r s i t y of nutritional requirements of bacterial collections suggest that i n t h e second s t e p - s e l e c t i o n process is
occurring
t o be p l a n t
depondent).
be
hyphal
(different
bacterial
negative bacteria
b y hyphae - a
(with
different
selection
r e q u i r e m e n t o f amino a c i d s
The i m p o r t a n t
process
in
recognition.
The
unique
structure)
wall
this
selection
seems
s t e p may of
supports
gramthis
hypothesis. REFERENCES
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S
Vancura, V., Orozco, MOO., Grau~va, O. and P~k~t, Z., 1989: Properties of bacteria in the hyphosphere of a veslcular-arbuscular mycorrhlzal fungus. Agrlc. Ecosystems Environ., 29: 421-427.