Antigenic similarity among Anabaena azollae separated from different species of Azolla

BIOCHEMICAL ANb BIOPHYSICAL RESEARCH COMMUNKATIONS

Vol. 109, No. 3, 1982 December 15, 1982

Pages

SIMILARITY AMONG ANABAEA’A AZOLLAESEPARATED FROM DIFFERENT SPECIES OF AZOLLA

ANTIGENIC

.I. K. Ladha Department Research Received

October

675-682

of Soil Institute,

26,

and I.

Watanabe

Microbiology, Los Baiios,

The International Laguna, Philippines

Rice

1982

SUMMARY: Direct fluorescent antibody (FA) reaction results of 5 FAs against symbiotic Anabaena azoZlae indicated that all the A. azoltae freshly separated from 32 specimens of AzoZZa collected worldwide (belonging to 6 different None of these FAs species) shared identical and highly specific antigens. exhibited cross-reaction with any of the free-living blue-green algae tested. FA absorption results confirmed these results and also indicate the existence of cross-reactive antigens between AzoZta leaves and the surfaces of A. azotlae. Antibodies made against free-living A. azoZZae did not cross-react with any of the symbiotic A. a.zotZae indicating either: (i) these isolates are not true isolates, or (ii) their antigenic properties were altered during isolation and culturing. Such possibilities and their implications are discussed. AzoZZa

is a genus

a nitrogen-fixing

blue-green

has recently

attracted

value

as green

plant

physiologists,

type

of symbiosis,

poorly

of aquatic

with

in rice

is

AzoZla has worldwide

and is

generally

symbiont are

several

isolation far

been

is

the

referred

of the fern's

increasing

interest

of

of AzoZZa-Anabaena symbiosis

compares

symbiotic

and infection

process

and is

A. caroliniana, The algal

systems.

belongs

to as Anabaena azotlae.

It

AzoZZa species

still

unexplained.

by six

recognizable

of

of the algal

symbiont,

Koch's

strains

to the Nostocaceae

is not

clear

and specimens

there

are

reports

postulates

isolated 00061291

675

Many aspects

A. mexicana, A. microphyZZa, symbiont

any of the algal

are

represented

Although

using

because

by this

of the symbiont.

and culturing

with

in N2 fixation

the other

same in the various

strains

satisfied

knowledge

associated

AzolZa-Anubaena symbiosis

Despite

distribution

and A. pinnata.

A. nilotica,

symbiotically

and agronomists

symbiont

A. filicuzoides,

Species:

cultivation.

known about

of the algal

is

of agronomists

microbiologists, the basic

which

Anabanea azollae.

alga

the attention

manure

what

the nature

fern

(for

whether or if

the

there

of the have not review,

so

see 1).

X/82/230675-08$01.00/0

Copyright 0 I982 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 109, No. 3, 1982 The most

BIOCHEMICAL

recent

report

is that

by Newton

and Hermen

Newton

and Hermen's

isolate

found

while

employing

The present antigenically

similar

of the world,

and

Anabaena species. implications

(2);

however,

antigenic

was undertaken

the symbiont

to see if

Data obtained

is

in

separated

antibody

in the AzoZZa specimens

(ii)

differences

and A. a.zoZZae freshly fluorescent

RESEARCH COMMUNICATIONS

of Anabaem from A. caroZiniam

of the isolation

indirect

study

AND BIOPHYSICAL

techniques

(i) obtained

similar

the algal from

to any of the

such a study

are

presented

between from AzoZZa were (3). symbiont

is

different

parts

free-living and their

considered.

MATERIALS

AND METHODS

The International Rice Research Institute (IRRI) AzoZZa culture collection contains 60 or more samples of AzoZZa spp. obtained worldwide. In the present study we used 32 strains of AzoZZa and several free-living bluegreen algae as described in Table 1. Free-living A. azoZZae isolated from AzoZZa caroZiniana was kindly provided by Dr. Newton, Peoria, Illinois, U.S.A., while A. azollae isolated from Azoi!la pinnuta and Anubaenu-free AzolZa pinnata were kindly provided by Mr. Bai Ke-Zhi, Beijing, China. AzoZZa cultures were maintained in mineral nutrient solution as described by Watanabe et aZ. AzoZla with Anabaena was grown on N2 as nitrogen source and Anabaena(4,5). free plants were grown on NaN03 (1 g B-l). The day temperature was 29OC and night temperature was 21°C, with average of 25'C at 30 klux for 12 hours. Blue-green algal cultures were grown in medium described by Allen and Stanier (6) with or without nitrogen source at continuous light intensity of 1000 lux and a temperature of 26 -+ 2'C. Anabaena filaments were separated from the log phase-grown fronds using the "gentle isolation method" but without using PVP-40 (7). Free-living blue-green algae were harvested at log phase. Cells of either free-living or symbiotic blue-green algae were washed twice in physiological saline and units per ml-based on haemocytoadjusted to about lo8 to log colony-forming meter counting. Fluorescent antibodies (FAs) were prepared as previously described (8). All FAs were titrated using twofold dilution steps and the staining reactions were carried out at dilution one step lower than that of the highest dilution The specificity of FA of all seven strains which gave maximum fluorescence. of A. azoZ7,ae was determined by using the controls laid down by Schmidt (9). Cross-absorptions were carried out by using a heavy washed suspension of the absorbing Anabaena strain in saline. For absorption with Anabaena-free AzoZla, the azolla plants (about 1 g fresh weight) were macerated in 1 ml saline in The cells and pestle and mortar and mixed with 1 ml of FA to be absorbed. FA were allowed to react for 3 h at 37OC and the supernatant recovered by centrifugation. This process was repeated with new cell suspension and finally the absorbed FA W8S recovered by centrifugation followed by filtration through a 0.2 pm-pore size membrane filter (Nucleopore Corp.). RESULTS AND DISCUSSION Five azotlae

fluorescent separated

antibodies from

4 species

(FAs)

were

of Azolla 676

prepared

against

and two FAs were

symbiotic prepared

A. against

WXHEMICAL

Vol. 109, No. 3, 1982 Table

1.

,4aoZZa

and

AND BIOPHYSICAL

blue-green

algal

Species

strains

Accession

RESEARCH COMMUNICATIONS

used

number,

in

the

present

designation

and

study.

source*

AZOLLA

A. pinnata

1: Bicol, Philippines; '2: Malaysia; 3: Bogor, Indonesia; 5: Bangkok, Thailand, 6: DAT (Department of Agriculture), Thailand; 11: Tangail, Bangladesh; 13: Khumaltar, Nepal; 15: Apalit, Philippines; 17: Vietnam Green I; 22: Tancheng, China; 23: Cuttack, India; 24: Floridablanca II, Philippines, 25: Ivory Coast; 35: IITA, Nigeria; 29: Changsha, China; 36: Cagayan, Philippines; 39: MIA, Australia; 44: Tamilnadu, India

A. filicutoides

101: 107:

East Walka

601:

Osaka,

A. mexicana

201:

California,

A. caro.?iniana

301:

Ohio,

A. microphylla

401:

Paraguay

A. nitotica

501:

Kosti,

A. fiZicuZoides

mbra

var.

A. pinnata

(Anabaena-free)

BLUE-GREEN

floe-aquae

ATCC 22664,

CCAP

A. oscilkrioides

CCAP 1403/11,

A. variabilis

Dr.

J.

408:

Guyana

1403/4

b,

J.

Uruguay

12;

412:

Paraguay

18

China

W. Newton,

Joseph

Dr.

A. de A.

Thomas,

Dr.

Joseph

Mr.

Bai

Ke-Zhi,

Mr.

Bai

Ke-Zhi,

USA

USA

Dr.

A. aeollae

Waard,

India

China

PCC 7120,

Dr.

R.

Rippka,

France

Anabaena

sp.

PCC 7122,

Dr.

R.

Rippka,

France

sp.

CA,

Dr.

C. Van

Baalen,

Dr.

Rippka,

PCC 73102,

Nostoc

sp.

Dr.

S. A. Martinez,

Gloeotrichia

sp.

Dr.

Oscillatoria

sp.

PCC 7515,

AzolZa

of

culture

a.zoZZae

A.

isolated

gave maximum

The peripheral

portion

the typical

vegetative

(4+)

Dr.

R.

Rippka,

described

red,

with

stained

their

cells. 677

that

corresponding cells

whereas

the

(5).

FA preparations

vegetative

of the FITC,

indicating

et al.

Watanabe

of AzoZZa.

red due to the autofluorescence

completely

Lanka

France by

2 species

fluorescence

color

France Sri

Philippines

are

from

USA

Kulasooriya,

of specifically

yellow-green

appeared

appeared

collection

Netherlands

China

sp.

sp.

The

India

Thomas,

Nostoc

The Netherlands

de Waard,

Anabaena

cells

1

y Tres,

Paraguay

Ke-Zhi,

W. Newton,

A. torulosa

7 strains

against

Treinta

Bai

Dr.

A. subcylindrica

*Details

cysts

202:

304: 1;

Mr.

Dr.

Anabaena

of the

USA;

Sudan

Tancheng,

A. cylindrica

with

USA

Japan

USA;

A. azoZZae

free-living

2, Germany; Cranmore,

ALGAE

Anabaena

all

Germany; 106: Hamburg Lake, N.V., USA; 108:

the

antigen.

fluoresced

central

portion

of chlorophyll. antibody

of

was produced

Heteromostly

of

dash (-) Underscoring

indicates indicates

species in Table

A. jZos-aquae

9

Other shown

(Bai

A. asoZZae

8

no

and 1

strains

22664)

3-k 4+

3+

* 3+

22

of

fluorescence. reaction.

-

3+ to 4+

reaction

algae

detectable a homologous

(ATCC

Ke-Zhi)

blue-green

(Newton)

1

no.)

AsoZZa

Free-living

strains in Table

(412)

A. microphyZZa

Other shown

(301)

A. caroZinium

A. aaoZZae

10

+'A

A. pinriuta

A. fi&uloides

(22)

(39)

A. pinnata

(106)

A. asoZZae

Symbiotic separated

from:

accession

Antigen

Immunofluorescence blue-green algae.%/

(species,

2.

7

Serial number

Table

4+

Numerals

3-k to

3+

3+

4+ 3+

4+

39

3+

azo2Zae

Immunofluorescence

Anabaena

l+

to

to

3+

5% 3+

4+

indicate

3+ to 4+

22 3+

3+

4-b

of

4+

301

3+

4+

reaction

selected

3+

106

against

3+

3+

3+

4+

412

and

minimum

to

G+ 3-k to 4+

accession

symbiotic

4+

4-f

1+ to

1+

1+

1+

1+

Newton

FA tested Bai

fluorescence.

-

free-living

maximum

and

4_+

-

Ke-Zhi

BIOCHEMICAL

Vol. 109, No. 3, 1982 Immunofluorescence free-living

the

reactions

blue-green

algae

A. azolZae

symbiotic species

and specimens

symbiotic

Nostoc

separated

(data

shown).

This

supported

22 FA with no longer

capable

with

with

free-living

any of the

all

the

blue-green

flos-aquae

strongly

Table

3.

with

of 5 species

3).

free-living

of Cycas

Anabaena from antigens.

This

Absorption

of anti-

A. azoZlae rendered Anabaem. (Newton)

the

FA

However,

did

not

remove the

against

two strains

FA against

A. azollae

symbiotic algae

of free-living

A. a.soZlae (Bai) or free-living showed

A. azollae and negative

reaction

cross-reacted

except with

reactivity of selected symbiotic to cross-absorbed FA of A. azollae

FA at similar and free-living

FA

Immunofluorescence 22

39

l+

to negative with

antibody

A. azoZZae

22.af

reaction

of 22 FA absorbed with

106

301

412

Newton 4-k

G+ 4+

-

-

-

--

4+

106

3+

-

-

-

--

3+

301

4-k

-

-

-

--

4+

412

3+

-

-

-

--

3+

are as in Table 2. 679

A. dilution.

22

Newton

all

A. flos-aquae (ATCC 22664).

Newton

not

blue-green

however,

tested

did

39

a'Footnotes

is

A. azollae.

A. azolZae (Newton),

None

any of the

specific

A. azollae

symbiotic

Antigen (accession no.)

Anabaenu of all

with

(Table

and

5 FAs against

the symbiotic

symbiotic

specificity.

FA against

tested.

that

results

prepared

exhibited

All

symbiotic

to be negative

roots

and highly

free-living

antibodies

A. azollae also

found

any of the symbiotic

of symbiotic

Fluorescent

reaction

identical

of staining

antigens

cross-react

indicates

or heterologous

of the FA with

specific

algae

clearly

also

several

symbiotic

but not

coralloid

by cross-absorption

homologous

absorption

tested,

the

2.

with

were

from

of AzoZZa share

species

further

cross-reacted

Cross-reactions

RESEARCH COMMUNICATIONS

FAs against

shown in Table

of AzolZa

algae.

all

of these

are

strongly

blue-green

not

AND BIOPHYSICAL

Vol. 109, No. 3, 1982 The close Newton

BIOCHEMICAL

identity

FA with

antibody,

of these either

whereas

Such results speculated either it

its

that

in modifying

essentially would

is

isolates

still

they

This

culturing.

and reintroduced

is

could

be clarified

into

Anabaena-free

revert

back its

I.e.,

nitrogen-free

dark

and nitrogen-free

free

leaf

medium

with

all

in antigenic

these

structure, of occurrence

low.

during

isolates

could

true

isolation

and

be reverted

back

specimens.

supplemented with

the A. azollae

to see if by growing with

in various

carbon

different

(Newton)

source

conditions

under

light

and

of AzolZa cell-

concentrations

extract.

antigens

When anti-22 suspension

characteristics

components of mutation,

of A. azollae are

their

if

antigenic

The frequency

isolates

properties

medium

The possibility surfaces

change

these

if

has little,

possibility

to be extremely

in attempts

antigenic

the

of already

the extensive

and the major

i.e.,

expected

have changed

to that from

We

A. azollae would

environment

to sporulate.

whether

or became mutated.

identical

we consider

mutation

We have not met success could

If

A. azollae

of symbiotic

that

shown).

The latter

symbiotic

Evidence

antigens

(10).

mutation unknown

or if

become

and ability

physiology,

of such spontaneous It

it

from any not

(3).

antigenicity

antigens

would

(data

et al. the

specific

structural

unchanged

antibody

of Gates

A. fzos-aquae.

be pleiotropic

morphology,

that

in Rhizobiwn indicates

any,

it

that

species

studies

then

homologous

of the

of homologous

A. azoZZae

separated

and surface

highly

serological

remain

freshly

when absorption

amount

and culturing,

morphology

free-living

effect

with

RESEARCH COMMUNICATIONS

was confirmed

no detectable

the

isolation

to such an extent

existing

remove

in agreement

unlikely

change

left by the

not

during

changed

think

strains,

did

are that

two strains

absorption

of Azolla

species

AND BIOPHYSICAL

that that

are

cross-reactive

FA and anti-412

FA were

of cross-reactive

A. azoZZae antigens

absorbed

(Table

between

with

680

leaves

was also

the crude

22, both

4) tested.

Azolla

have on their

AzoZla cells

with

AzoZla pinmta

of Anabaena-free symbiotic

A. azollae cells

the symbiotic

This

leaf

extract

the FA reduced indicates

and the

surfaces

examined.

reactivity

the existence of A.

Vol. 109, No. 3, 1982 Table

4.

BtOCHEMtCAL

AND BIOPHYSICAL

FA reactivity of symbiotic Ambaem azoZkze to cross-absorbe FA of A. azolke strains 22 and 412 with Ambcena-free AzolZa.~3

Immmofluorescence Antigen (accession

Unabsorbed 22

412

22

32

4+

39

3+

3+

106

3+

3+

301

3+

3-k

412

4+

-4+

are

as in

Table

The possibility

by absorbing

AzolZa. out

proposed

that

This

It

I+

not

antigens

with

the host

demonstrated algal

cells

cells

rather

If

means was also

FA with

the leaf

checked

extract

of

of A. asollae (Bai)

the reactivity

identified

requires

and the lectins

be expected

process

always

present

plant

during

that

It

an interaction

symbiosis that

studies

of other the

This

(14).

thin-walled

681

of

been reported

on the role

symbiont's

cycle

continuity

these

The presence

mechanism

blue-green

and transmission are

is also

could

exist

of cross-

are needed.

in AzoZta-Anabaena symbiosis

in the megasporocarp, the sexual

(11)

between

has recently

a similar

symbiont

symbiosis,

in the megasporocarp spores.

symbiosis.

of the legumes.

Further

of algal

by means of light

than

as polysaccharide

in AzoZZa-Ambaem symbiosis

from

In AzoZZa-Anabaena are

1+

and Rhizobim

legumes

and lectins

to be different

spores?)

reduce

symbiosis

therefore

The infection

(13).

(Bai)

were

in the AzoZla-Anabaenu symbiosis.

appears

1+

by nonspecific

in the Azolla-Ambaena

could

reactive

1+

1+

which

between

antigens

(12) *

412

2.

did

a successful

cross-reactive activity

AncLbaenu-free AzoZZa

22

A. a.zolZae

antigens

reported

lectin

reaction FA with

the possibility.

Cross-reactive have been

Absorbed

of absorption

the free-living

Anabaenu-free FA ruling

FA

no.)

a/Footnotes

azollae.

RESEARCH COMMUNlCATlONS

algal

symbiotic

vegetative

cells

maintaining (14)

has

microscopy

and resemble

of association

(or

association

Becking electron

systems

also

that

vegetative

between

algal

the

BIOCHEMICAL

Vol. 109, No. 3, 1982 symbiont

and host

AND BIOPHYSICAL

would

seem to rule

by free-living

Ambaem.

Furthermore,

algal

is unlikely

symbiont

to exist

out

the need it

suggests

in the

free-living

RESEARCH COMMUNICATIONS for that

infection

of the host

such a host-specific state.

ACKNLOWLEDGMENTS We thank Dr. K. Fowler (Portsmouth, UK) for his valuable suggestions and comments on the manuscript. We also thank Drs. J. W. Newton (USA), A. de Waard (The Netherlands), Joseph Thomas (India), R. Rippka (France), Van Baalen (USA), M. Martinez (Philippines), S. A. Kulasooriya (Sri Lanka), and Mr. Bai Ke-zhi (China) for providing us blue-green algal cultures and Professor J. V. Pancho and Professor R. Aspiras (University of the Philippines at Los Baiios) for their help in getting coralloid roots of Cyeas. This work was supported by the United Nations Development Programme fund. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Peters, G. A., Mayne, B. C., Ray, R. B., and Toia, R. E. (1979) In: Nitrogen and Rice pp. 325-344. IRRI Symposium, Los Baiios, Philippines. Newton, J. W., and Hermen, A. I. (1979) Arch. Microbial. 120, 161-165. Gates, J. E., Fisher, R. W., Goggin, T. W., and Azolan, N. I. (1980) Arch Microbial. 128, 126-129. Watanabe, I., Espinas, C. R., Berja, N. S., and Alimagno, B. V. (1977) IRRI Research Paper Series No. 11. Watanabe, I., Bai Ke-zhi, Berja, N. S., Espinas, C. R., Ito, O., and Subudhi, B. P. R. (1981) IRRI Research Paper Series No. 69. Allen, M. M., and Stanier, R. Y. (1968) J. Gen. Microbial. 51, 203-209. Peters, G. A., and Mayne, B. C. (1974) Plant Physiol. 53, 813-819. Ladha, J. K., Barraquio, W. L., and Watanabe, I. (1982) Can. J. Microbial. 28, 478-485. Schmidt, E. L. (1973) Bull. Ecol. Res. Commun. (Stockholm), 27, 67-76. Bohlool, B. B., and Schmidt, E. L. (1980) In: Advances in MfcrobiaZ Ecology (Alexander, M., ed.) pp. 203-241, Plenum Publishing Corp. D. H. (1975) Appl. Microbial. 30, 1017-1033. Dazzo, F. B., and Hubbell, Mellor, R. B., Gadd, G. M., Rowell, P., and Stewart, W. D. P. (1981) Biochemical Biophysical Res. Commun. 99, 1348-1353. Stewart, W. D. P., Rowell, P., and Rai, A. N. (1980) In: Nitrogen Fixation (Stewart, W. D. P., and Gallon, J. R. ed.). pp. 239-277 Academic Press. Becking, J. H. (1978) Ecol. Bull. (Stockholm) 26, 258-273.

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