Properties of bacteria in the hyphosphere of a vesicular-arbuscular mycorrhizal fungus

Properties of bacteria in the hyphosphere of a vesicular-arbuscular mycorrhizal fungus

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 ...

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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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F. gunc (Editors). Soii microbial associations. Control of structures and functions . Academia and Elsevier, Prague and Amsterdam, pp. 57-144. Vuurde, J.W.L. van, Kruyswyk, C.J. and Schippers, B., Bacterial colonization of wheat roots in a root-soil

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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.