"

1 , .... . " ;-*. , 4 , ... \,.

Stu

.' "

ABSTRACT

... Ph.D. Maurice Lalonde MICROBlOLOGY

rStudie~ of the Alnus ~rispa var. mollis Fern.

Root Nodule Symbiosis.

The thin, filamentous, branched and septate root n~du!~ endophyte of

Alnu8 crispa var~ mollis Fern. exhibited a prokaryotic cytology. The actino-

mycete-like endophyte penetrated through the host celi wall and became envelo-

ped by a ~apsular material, the whole being enclosed by the host membrane.

After spreading in the cortical cells, the hyphae started to produce septate

vesicles near the hast cell wall. Endophyte ghost forms were observed at the

base of th root nodule.

Free cytoplasmic multivesicular bodies fused with the membrane enve-. , lope that surrounded the endophyte cells and tiberated an electron dense mate-

rial. The resulting endophyte capsule was mainly composed of granular and

fibrillar non-sulfated and de-esterified polygalacturonic acid or its salts.

This pectic nature of the endophyte capsule also demonstrated with other non-

l 1-eguminous root nodule endophytes.

Two filamentous, branched and septate dctinomycetes were/1solated

from root nodules and cultivated on a chemically defined medium. These two

homol~gous isolate~ were morphologically similar to the nodule endophyte, but

were unable ta nodulate the host plant. Immunolabelling reactions demonstra-

ted the homology of 'the avirulent isolates with the nodu~ endophyte. The LL

and meso-isomers of diaminopimelic acid were present in similar proportions

in the nodule endophyte and nodule isolates. ~

.. -

o

,j ! c

l '

\) RESUME

~h.D. Maurice Laloriae Microbiologre ..

L'endophyte nodulaire d'AZnus arispa var. mollis,'qui est mince,

filamenteux~ ramifi et sept, exhibe une cytologie procaryotique., L'endophyte . , qui ressemble un actinomyc9te, pntre travers les parois cellulaires de

l'hte et devient envelopp par un matriel capsulaire, le tout t~nt entour

par une membrane hte. Aprs leur dispersion dans les cellules cortLcals,

les hyphes commencent produire des vsicules septes prs de la paroi cellu-

laire hte. Des formes fantmes de l'endophyte sont observes la base du

nodule racinaire.

Des corps multivsiculs qui sont libres dans le cytoplasme de la

cellule hte, se fusionnent avec l'enveloppe membraneuse qui entoure les

formes endophytiques et librent un matri,el relativement opaque aux lectrons.

La capsule endophytique qui en rsu1te, est principalement compose de granules ~

et de fibrilles non-sulfates et ds-estrifies de l'acide polygalacturoniqu~

ou de ses sels. Cette nature pectique de la capsule endophytique est aussi

dmontre chez les endophytes de~d' autres non-lgumineuses nodules.

Deux actino~yctes filamenteux, ramifis et septs, ont t ~sols

de nodules racinaires et, cultivs sur un milieu de composition chimique dter-

mine. Ces deux isolats qui sont homologues, ont une morphologie similaire

celle de l'endophyte du nodule, mais sont incapables de noduler la planie hte. "

Des ractions d'immuno-marquage dmontrent une homologie des isolats avirulents l

avec l'endophyte du nodule. Les isomre~ LL et meso de l'acide diaminop~mlique

sont prsents en quantits similaires dans l'endophyte du nodule et dans les

isolats nodulaires.

, (

c - -

j

/

. ,

r --r

If

\,

t ..

, ' . ,/

Suggested short title

o

Alnus root nodul~ endophyte.

..

) r

Maurice Lalonde .,

f)

..

, ...

----

,f

/ ..

(

---,-__ --- - --KCKNOWLEDGEMENT

.. l wish to express- my' sincere gratitude to Dr. R. Knowles, Professor

."

il- of the Department of Microbiology, Macdonald College, for his interE!$t and

guidance throughout this investigation.

~ thanks are extended to staff and student members of the Micro-

biology Department for their kind help and encouragements' during these studies.

The author is grateful for the facilities made available to him by Dr. J.F.

Peterson of the Department of Plant Pathology. Thanks are also extended to ,

Prof. C.E. Tanner of the Institute of Parasitology, for his assistance with

the cardiac bleeding of the immune rabbits.

Sincere appreciation is extended to Mr. J. E. Gilchrist for the hell'ing

hand at the electron microscope. l am grateful t~'MiSS K. Roche, Mrs L. V. Warner-Roche and Mr. J. Brasseur for.their very ki~. help in preparing this manusc ript. l also wish to thank 11iss. P. Lalonde for skilfully typing the

manuscript.

/ The author i8 indebted to the N .R.C. of Canada for the award of a

N.R.C. Postgraduat~ Scholarship, to McGill University for a McConnell.Memorial

Fellowship and the Department of Education of Quebec for a Graduate .scholarship .

.. o

vi .

\

)

CLAIM OF CONTRIBUTION TO KNOWLEDGE

1. The u1trastructure of the ALnus apispa var. moLLis Fern. root nodule

endophyte is describd and suggests an actinomycetal nature.

2. The insoluble, granular and flbrillar capsule of the endophyte is

demonstrated to be most1y composed of polygalacturonic acid or its sa1ts.

3. A possible mechanism for the host biogenesis of the endophyte F~ctic , capsule 15 proposed.

4. The' pectic nature of the endophyte capsule iS,extended to 12 other CI

species of non-1eguminoua ~ot nodule symbiosis.

5. Immuno-labe1s that specifica1ly recognize the nodule endophyte are

obtalned.

6. Immunolabe11ing techni~ues demonstrate the endophytic nature of two

avirulent root nodule lso1ates. (

, ! .

7. Whole cell hydrolysates analysis demonstrate the presence of both LL {

and meso-DAP lsomers in the root nodule endophyte,and nodule isolates.

'j. vii

..

1

\"I~'~1. , . ,

"-

-

'\

n

TABLE OF CONTENTS

DEDICATION --

ABSTRACT

RESU~E

ACKNOWLEDGEHENTS ,-. CLAIM OF CONTRIBUTION TO KNOWLEDGE

TABLE oy CONTENTS LIST OF TABLES

LIST OF FIGURES

1.

2.

3.

3.l.

3.2.

3.3. ,

'3-.~. -\

4.

4.l.

4.2.

4.2.l. 4.2.2. 4.2.3. 4.2.4. 4.2.4.l. 4.2.4.1.l. 4.2.4.1.2. 4.2.4.2'. 4.2.4.2.1. 4.2.4.2.2. 4.2.4.3. 4.2.4.4. 4.2.4.5. 4.2.4.6. 4.2.4.7. 4.2.4~8. 4.2.5. 4.2.6. 4.2.7.

GENERAL INTRODUCTION

REVIEW OF LITERATURE

ULTRASTRUCTURE OF THE ROOT NODULE ENDOPHYTE

INTRODUCTION

MAlERIALS AND METHODS .' .~" ~ S~ESULTS '

1)

DI~USSION

ULTRAS'TRU:JE, COMPOSITION AND OF THE ENDOPHYTE CAPSULE

INTRODUCTION i

MATERIALS AND METHODS

BIOGENESIS

Hand eut sections of root nodules Uranyl~lead ~tained nodule sections Purified endophyte-suspension Polysaccharide'staining tests Alcian blue Light microscopy Electron microscopy Calcofluor staining Smears Nodule sections PAS reaction IKI-H2S0~ reaction Aniline blue r Ruthenium red Ruthenium red-Os0

4 Hydrokylamine-ferric chloride reaction Enzymatic degradation of the endophyte capsule Immunoferritin method Solubilization test of the endophyte-capsule

viii

PAGE

il

Hi

iv

vi

vii

viii

xi

xii

1

3

9

, ,

l ,

\

4.3.

4.3.1.

4.3.2.

4.3.3.

4.3.4.

4.3.5. 4.3.6. 4.3.7. 4.3.8. 4.3.9.

4.3.10.

4.3.11. 4.3.12. 4.3.13.

'4.3.14.

4.3 :s1. 4.3.~.

4.4"

5.

5 .1.

., 5.2.

5.2.1. ..2.2. 5.2.3. 5.2.4. 5.2.5. 5.2.6. 5.2.7.

5.2.8. 5.2.9. 5.2.10. 5.2.11. 5.2.12.

5.3.

5.3.1. -'" 5.3.2. 5.3.3. 5.3.4. s.~.s.

, " ..! 1-

RESULTS

Electron microscopy of ~ranyl-lead stained nodule sec tions.

Electron microscopy of alcian blue stained nodule tissues.

Fluorescence microscopy of calcofluor stained no-dule tissues.

Periodic acid-Schi,f's (PAS) reactfon on nodule se(!tions .

Cellulo~e staining nodule sections. Callose staining of ule sect~so Alcian blue sta nodule sections. Ruthnium red staining of nodule sections. , . EM observations of RR-Os0

4 stained endophyte-sus-

pension. Hydroxylamine-ferric chloride reaction of nodule sections. In~lubility of the endophyte capsule. Enzymatic degradation of the endophyte capsule. Thin layer chfomatography of the capsule degrada-tion products.

Hogt cell wall localization by immunoferritin labe11ing.

Biogenesis of the endophyte capsule. Capsule of other non-Ieguminous nodule endophyte.

DISCUSSION

ISOLATION OF AVIRULENT ROOT NODULE El\TJ)OPHYTE

INTRODUCTION

MATERIALS AND METHODS

Isolation of field nodule endophyte. Isolation ofaxenic nodule endophyte. Subculture of the nodule isolates. Nodulation tests. Preparation of specifie gamma globuline Preparation of FITC-conjugated gamma globuline Staining procedure and examination of stained materials.

Dilution aftd adsorption of conjugates. Whole celi DAP analysis. Whole ce~ sugars analysis. Lysosyme resistance. Catalase pro~uction.

RESULTS

Field nodule isolate. Axenic nodule isolate.

( -

Cultural characteristics of the nodule isolates. Assay of nodulation. Reactions of isolates FA conjugates with A. arispa var. molli8 endophyte.'

ix "

-

PAGE

37

37

38

38

39

39 , 39 39 4,2 42

43

43 45 46

46

47 48

63

68

68

69

69 70 73 73 74 75 75

75 76 76

_'" 7 7 77

77

77 78 79 80 80

..

, 5-.3.6.

5.3.7.

5.3.8.

5.3.9.

5.4.

fi.

7.

8 ..

9.

1

,

:\ Reactions of isola~es FA conjugates with known species of actinomycetes. ~actions of isolates FA conjugates with Alnus endophytes.

Reactions of isolates fe~ritin conjugates with A. crispa var. mollis endophyte.

Whole cel! hydrolysates. '

DISCUSSION , ,

GENERAL DISCUSSION

CONCLUSION

SUMMARY

REFERENCES

"

x

()

. '\ ,

PAGt 82

82

86

87

94

99

~ 102

103 L

105

-

LIST OF TABLES

)J, 1

l ALnus root nodule investigation protocOI.~

II PAS reaction of A. ~ri8pa var., mOll~Hle sections.

III ~cian blue staining of A. ~ri.8pa var. mollis root nodule

sections . .,..

IV McCready-Rkeve's test for pectic substances.

V McCready-Reeve's Pectin test of A. cri8pa var. mollis

nodule sections.

J

VI Non-leguminous root nodules tested for their endophyte

VII

VIII

x

capsule.

Composition of the Harvey Special medium.

Immunofluorescent reactions of A. crispa var. mollis nodule

endophyte stained with FA conjug~tes of nodule isolates.

Immunofluorescent reactions of a range-of known speci:es of

actinomycetes stained with FA conjugates of the nodule

isolates.

z, Immunofluorescent reactions of Alnus nodule endophytes

stained with FA conjugates of nodule isolates,

!

XI Indirect immunoferritin reactions of A. crispa var. mollis .. nodule endophyte stained with ferritin conjugates.

" .'

xi

PAGE

8 ..

, 40

4'1

50

72

81

83

85

88

Q

1.

2."""

3. '

4.

5.

6.

7. 1 {

8.

9.

10.

11.

. 12.

(.

" LIST OF FIGURES

, Ultra-thin s~ction of nodule tissu~ showing a

cell newly infected by the hyphal form of the endophyte.

, . , A host cortical celi infected oy hyphae and by the vesicular ,

fBrm of the endophyte.

A host cortical cell containing a high density of hyphae in

its center, and septate vesicles near its cell wall.

A hast cell nu~leus surro~nded by hyphae and vesicles.

Detail of figure '4.' showing two hyphae surrounded by the , ' nucleoplasm of a host cell nucleus~ , . ,

An other view of a host cell nucleus surrounded by hyphae

and vesicles.

Along side the hyphae, a zone of host CytoPla~""1thOWS , '\ mitochondria and small starch grain ~ontainJ' in, ~"n

amyloplast. . L Nearby the hyphae. a zone of host cytoplasm shows a

mitochondria, ribosomes and ~ dictyosome.

Longitudinal and transversal sections of hyp~e in a newly"

infected host cortical cell.

Detail of figure 9, showing the ramification of the hypha.

Another view of me~somes in young hyphae.

Hypha showing septations. '}

xii

P4GE

13

13

14

14

14

15

15

15

16

_ 16

16

17

13.

14.

15.

16.

Detail of a hyphal septation.

Transversal section of hyphae showing a tubular mesosom. 't

Transversal section of hyphae showing a tubular mesosome.

Longitudinal section of a young hypha showing granules in

its cytoplasm.

17-21. Penetration of hyphae through the host celi waiis.

22: The tip of a parental hypha beginning to swe11.

23. A young v~icle adjacent to the host cell wall.

24. Ultra-thin section showing theocontinuity of the capsu1ar ~-,

Adjacent to the ~ust cell wall, a young vesicle with a r ; . .

complete septation shows a side septation in formation.

A high1y septate maturing vesicle, showing the formation

of side septations from completed septa.

xiii

PAGE

17

17

17

18

19

20

20

20

20

21

21

22

22

23

24

32.

33.

34.

35.

36.

'37.

38.

39.

40.

41.

1

4.2.

43.

44.

=

t ...

High magnification of a crystalline inclusion.

Detail of a mature vesicle showing the continuity of the

septation with the cell wall.

Electron micrograph showing a lamellar mesosome being

produced by the invagination of the cytoplasmic membrane.

Ultra-thin section illustrating a tubular mesosome still

atta~hed to thb tip of a vesicle septum.

An infected host cortical cell in the basal. part of the

nodule.

Septate dense vesicle.

Micrograph demonstrating the ~esorption of the endophyte

cytoplasm.

Micrograph demonstrating that the endophyte ghost~ forms

are not the result of a fixation or embedment artefact.

High magnificat ion of the ghost hyphal and ghost vesicular

forms of the endophyte.

Electron micrograph showing the penetration of the hast "

cell wall.

Detail of figure 41, showing the c'on~inuity of the hast cell wall material with the so-called endophyte capsular

material. J

/

Electron micrograph showing the penetr,ation of the hast

cell wall by an endophyte hypha.

Longitudinal section of a hypha crossing the cell wall of

a host infected cortical cell.

xiv

PAGE

24

25

26

26

27

27

28

- 28

28

51

51

51

52

".4 '

c ,

-

45.

46.

47 "

Detail of figure 44, ~howi~~ the continuity of the hyphal

capsular material with the host cell wall.

Trans~sal section of hyphae agglomerated i~ the~host

cell wall.

,. J.~ , l.. \

Detail of figure 46, showing the fibrillar structure'cf

" the aician blue stained capsular material surrounding

the hyphae.

48,

49,

50.

51.

52.

53.

54~

55.

Incident-light fluorescence micro~copy of host cortical

cells in~ed by the root nodule endophyte, after cal-

cofluor staining.

The same microscopie field shown in figure 48 but photo-

graphed in phase contrast microscopy.

Epon section of a calcofluor-stained root nodule showing

the early stages of infection.

Ghost vesicles and ghost hyphae showing ~lysaccharidepositive reaction with the calcofluor stain.

, PAS reaction on an Epon section of a formaldehyde-fixed

-root nodule.

McCready-Reeve's pectin reaction on a hand c~t section of

a fresh root nodule after over-night esterification in

acid-methanol. '

Endophyte vesicle showing its capsule strongly stained by

the ruthenium red-Os04

mixture.

~~tail of a vesicle capsule after its staining with the

ruthenium red-Os04 stain.

xv

PAGE

52

52

52

53

, )

53

54

54 .

54

54

55

55

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

Another view of an endophyte vesicle stained with the

ruthenium red-Os04 mixture.

Unstained section of an endophyte-suspension treated with

a cellulase-hemicellulase mixture and glutaraldehyde-

.osmium fixed.

Ultra-thin section showing the hyphal endophyte capsu~e

labelled with a specifie immuno-ferritin complex after

the capsule solubilization test.

Endophyte-suspension treated with a pectinase solution.

Endophyte~suspension treated with a pectinase solution.

~

A partially pectinase-degraded vesicle capsule, jained

with the ruthenium red-Os04 mixture. J An endophyte-suspension treated for 24 hours with a

pectinase solution, and stained with a ruthenium

red-Os04 mixture.

Endophyte-suspension, previously treated with pectinase,

exposed to anti-isolate whole cells immunoglobulin and

to ferritin-labelled goat anti-rabbit immunoglobuline

M Detail of an endophyte vesicle treated as in figure 63.

Ultra-thin section of glutaraldehyde-OsO~-fixed and

uranyl-Iead-stained root nodule tissue.

66-67. Formation of void areas by the shrinkage of the vesicle

cytoplasm.

68. Free multivesicular body in the ytdplasm of a 000-

infected host cortical celle

PAGE

55

55

56

56

56

57

57

58

58

58

59

60

69.

70.

71.

72. s __

, r

1 73.'-

."~!.-

74.

75.

76.

77.

78.

79.

..

Lomasome-type paramural body resulting from th~ fusion of

a free multivesicular body with the plasmalemma.

Adjacent to two endophyte hyphae, a free multivesicular body

is seen in the host celi cytopla$m.

A zone of host cell cytoplasm showing a mitochondria, dictyo-

some and secretory v~sicles at the tips of cisternae.

Adjacent to an endophyte vesicle a free'multivesicular body

iS,fusing with the host plasmalemma.

Between the host plasmalemma and the capsule of an endophyte Il'

vesicle, electron debse vesicles are seen liberating their

electron dense material.

Vesicles of paramural bodies are seen near the capsule of an

endophyte vesicle.

Resulting from the fuSion of a free multivesicular body with

the host plasmalemma, small vesicles are in direct contact , with the capsule of a hypha.

Two clusters of small vesicles, forming lomasome-type para-

mural bodies, are seen adjasent to the capsule of an endo-

phyte vesicle.

Detail of figure 76, showing a cluster of small vesicles

situate between the host plasmalemma and the vesicle capsule.

-2 Fi~ld nodule isolate ob6erved in a 10 suspension dilution

tube following 150 days of incubation at 4C.

A host cell, that appears,to be alive, is surrounded 'by the

filamentous endophyte-like field nodule isolate.

PAGE.

60

60

60

61

61

61

61 ,

62

62

89

89

, 1 80. 81.

82.

Old in vivo-produced vesicle showing the shrunken contents

and aalencapsulated parental hrpha.

Field nodule isolate after its first transfr to Harvey

Special medium.

Field nodule isolate following its first transfer to Harvey

Special medium.

83. Field nodule isolate showing septate vesicles and numerOU8 -- - --------

particules produced by the fragmentation of septate hyphae .

. - ... 1

84.

85.

36.

87.

88.

89.

90.

Hicrocolonies of the field noqub.e isolate observed after

2 weeks growth on Harvey Special agar .

. Axenic nodule isozate grown in liquid Harvey S~ccial aedium

for 3 weeks under anaerobic conditions.

- -------

Field nodule isolate grown in liquid Harvey Special medium

, for 8 weeks under anaerobic conditions.

Electron micrograph of a diphtheroid fom of the field

nodule isolate.

Inident-light fluorescence microscopy of a mixture of field

and axenic nodule isolateC

cells, Chthi'n an A. -cl'ispa var.

moUi& endophyte-sl.lspension, stained with field nodule F1 conjugate.

Electron micrograph of a field nodule whole cell reacting

with specifie isolate rabbit antibody and stained with

ferritin-Iabelled goat anti-rabbit gamma globulin.

Ultr~-thin section illustrating the isolate immuno-ferritin

complex adhering to the nodule isola~e celi wall.

xviii

PAGE

89

89

89 '-

'89 ,

90

90

90

90

91

92

92

91.

93. ,

94.

95.

96.

97.

1

~

Mixture of isolate whole cells'reacting w~th normal rabbit

serum and st~ined with ferritin-Iaber'ld loat anti-rabbit

gamma globuline

Mixture of isolate cells in an endophyte-suspension after

reaction with endophyte-susp,ension rabbit antibody and . staining with ferritin-Iabelled goat anti-rabbit~gamma

globuline

Detail of an enca~sulated endophyte vesicle specifically

stained with the endophyte-suspension Umnuno-f~rritin

complexe

Mixture of isolate cells in an endophyte-suspension, stained

with specific isolate Umnuno-ferritin cOmplexe ~

Endophyte-suspension treated with a peetinase solution in

the absence of antibiotic and stained with the isolate

immuno-ferritin comple~

Detail of a pectinase-treated vesicle stained with the

specifie isolate immuno-ferritin complexe

1 Pectinase-treated endoph~~ bypha stained with the specific

isolate immuno-ferritin complexe

\,

1

xix

PAGE 0

92

92

92 '

93

93

93

9~

i !' \

(' l

e.

..

_ r

-----. __ J-----

1. INTRODUCTION

~ The demon$~ation of the nitrogen-tixing root nodule symbiosis as

shown among a considerable number of non-1eguinous angiosperms is obviously \

of some interest. This symbiotic system pennits ,significant contributions to

th& nitl"pgen economy of a numb~.r ,o~ ecosystems. The importance 01 Alnus and

Dryas in primary plant succession in the Glacier Bay area of Alaska has been

demonstrated by Cracker and Major (1955), and Lawrence et al (1967). Not on1y

are non-leguminous plants important for improving the growth of associated

plants (Tarrant and' Hiller, 1963; Wollum II and Youngberg, 1964; Daly, 1966 ;

Franklln aod Pechanec,1968; Newton et aZ,1968; Akkermans,1971; Tarrant and

Trappe,1971; Uemu~,197l; and Fessenden et aZ,1973) but it h.~s also been re-

ported that they improve the productivity of lakes (Goldman,196l).

q

Richard (1971) in his palynological inve~tigations of Late-Pleisto-. ,~. cene s~diments~ :Elf southern Quebec, noted the occurence of the pollen of .

, . Shephel'dia canz.q.ensis, Myrica gale, A lnus rugosa and A lnus arispa, wl~ich de-

monstrated the presence of these non-l~guminous nodulated plants following the

last glacial retreat. From~personai observations,' it appears tnat Alnu$ crispa

var. mollis Fern. i6 still very abundant on sandy soils or eroded slopes along

the Saguenay river and in the Lake St. John area. In aIl of the root systems

of this green silky aIder e~amined, extensive nodulation was observed. This k

occurence of well-nodulatecl A. crispa var. mollis, usually on poor 60ils,

suggests that, as for the other nodulated non-leguminous angiosperms, it could

play a signfficant roie. in the over-ail nitrogen economy of the unsettled area.

Ecological research i8 required to tbrov ~ght on the nitrogen fixa-, J

tion capacity of this aIder species in the f~eld. The potential Q~ ~his

symbioS!s i5 now being studied by the Dr. J.A. Fortin's research group at

Laval Universit~, Qubec. In addition, fund~ental knowledge of this symbio-

-----

sis might, allow similar manipulation of the uodulation as is done with the "

legume-Rhizobium nitrogen-fixing root nod~SymbiOSiS. Unfortunately, iso- ~ !~ lat ion and i~ification of the causal organisms of the 'nitrogen-fixing root . ".,~ nodule of non-leguminous angiosp~S had not been achieved. Consequently, the

knowledge of this non-Iegume symbiosis will "remain inferior to that provided

, by the legumes ,50 long as pure cultures of the endophytes remain unavailable"

, . -

"

2 -

(Bond,1970). 50 the pr\mary objective of my investigation was to isolate the

root nodule endophyte of A. cri8pa var. mollis.

,; The results obtained are presented in three parts. The first part

describes the ultrastructure of the root nod~le endophyte. The second part investigates the endophyte capsule, and the last part deals with two actino-

mycetal strains, isolated from field-col~cted and from 8~enic in vi~roproduced root nodules, in ord,er to demonstrate their endophytic nature. Of

special interest here 'is the prnblem of how to telate an avirulent endophyte

observed root nodule endophyte. The progression of this

described at the end of the review section.

-'

1

/

'-

o ('

7 7

- 3 -

2. REVIEW OF LITERATURE

The bioehemieal aspects of the nitrogen fixation has been reviewed

reeently in considerable detail (Hardy and Burns,1968; Hardy et at,197l;

Dalton and Hortenson,l972; Postgate,1972; Di1worth,1974). The genetie aspects

of the nitrogen fixation has been reviewed by Streiche;--arut--Valmtine (1973).

The recent acetylene-ethylene, asssy, which replaces the bioassa~~r the incor-

poration of stable isntopa nitrogen-15 in order to demonstrate and measuie a ,

nitrogen-fixing abi1ity, has been reviewed by Hardy et aZ (1968, 1973). f~" ~

Becking (19~6), Stewart (1966, 1967), Bond (1967a, 1967b, 1971), l1'1shustin and

Shil 'riikova (1971) and Silvester '(1974) have discussed the role of nitroge~,

fixing plants.

The fo11owing review and discussion will be confined ta the liter-

ture on the nitrogen fixing non-leguminous angiosperms with emphasis on the

attempts to isolate the root nodule endophytes.

The presence 9f~the nitrogen fixing root nodule with non-leguminous

angiosperms has been found ~fn more than ten genera throughout the wor1d (Bond, ; ~

1968; Becking,1970a; Si1ver,197l). It has been'proposed that the site of the

nitrogen fixation is within the endophyte cells eontained in the host infected

cortical cel1s of the toot nodule (Akkermans,l97l).

On a morphologie al basis, the root nodule endophyte has been associa-

ted ,with fungi (llawker and Fraymouth,19Sl). But according to recent light and

electron microscoyy studies, the prokaryntic, branched, septate and filamentous

endophyte was identified as an actinomycete (Fletcher,1955; Furman,l959;

Lalonde and Fortin,1973; Silver,1964; Becking et ~,1964; Gardner,1965; Gatner

and Gardner, 1970), After the spreading of the thin and septate hyphae in the

host cortical cells, there was a swel1ing of the api~es of filaments wit~ the

formation of the so-called vesicles (Gardner and Gatner,l973). These vesicular ...

protuberances aI 50 occuied in free-living actinomycete un~er special cultural

conditions (Lechevalier and Lechevalier,1969). In the basal parts of the root

nodule, bacteria-like cells were sometimes seen (Becking et~Z,1964). It was

suggested that they had been formed by the fragmentation of the endophyte hyphae

(KHppel and W8rtenberg,1958). The breakin~ up of filaments into pieces occured

- t

$ ?

- 4 -

in, free-living actinomycetes (Lechevalier and Lechevalier, 1967). , "

Despite many claims to the contrary, isolation, subculture and , nodulatif:>n tests of the aseptic host plant ~ith iso1ated endophyte has not

been achieved. The literature pertaining to the isolation of micro-organisms

from non-1eguminous root, nodules has been revie~d by\A11en and Allen (1958,

1965), Becking (1968,1970a), Bond (1959,1963,1967a) antl Uemura (1964). ~ r

Pek10 (191,0) was the first worker to claim the isolation of the

endophyte of A Znu8 gl,ut'inosa and called it Actinanyces aZni. A1beit no re-

infection of the host plant was obtained.

Since that time, many workers have tried to isolate the causal

organism of the root nodule but they did not succeed in providing definite

proof by re-infecting the host plant (Lieske,1921; Krebber,1932; Bou~ens,1943;

Quispel,1954a,1'954b,1955,1958,1960; Uemura,1952a,1952b,1961,1964,'1971;

F1etcher,1955; Silver,1964; Allen et aZ,1966; W~ollum II et at, 1966; Becking,

1970a, Ydbngberg and Hu,1972).

Von Plotho (1941) claimed successful infection experiments with a

strafn iso1ated from AlnuB root nodule. But Bouwens (1~43), Quispe1 (1954a),

Pommer (1956), Taubert (1956) and Becking (1970a), could not carry out a re-

infection with the same strain. Pommer (1959) isolated an actinomycete from

sections of a surface-sterilized Alnu8 root nodule. Following itsin vitro

growth on glucose asparagine agar, this 'isolate produced septate vesic1es at

the t1p of the hyphae which gave rise to bacteria-like cells. Furthermore,

this actinomyceta1 isolate induced the nodu1ation of aseptic host plants.

Unfortunate1y, Quispe1 (1960) and Uemura (1964) fai1ed to confirm Pommer's

findings.

Fiuczek (1959), Niewiarowska (1961) and later Dani1ewicz (1965)

reported the isolation of root nodule endophytes, but their findings are still

n~t confirmed. Koslova e't al (1966) iso1ated bacterial strains from the nodules of' sea buckthorn (HippophaM rhamnoides L.). These iso1ates were simi-

lar in their properties to the genus Rhizobium and were able to induce the

nodu1ation of the host plant. These surprising results are not yet confirmed.

2

, -,

"

--

P 7

"~

- 5 -

... " :,) Quispel (1960) believed that the endophyte might bave special

nutrienl requirements and he reported'that a lipid extract from Alnus roots '(

promoted the maintenance of the virulence of~an endophyte culture. Becklng

(1965) obtained tissue cultures of aIder root nodules containing the endophyte.

However. he did not ob tain the transmission of the endophyt'e from the infected

cells ta newly formed cal1us tissue. Becking (i970b) speculated that the

causal organisms of the non-leguminous root nodules are &bliga~~ symbionts and

c1assified them, on a morphological. b.asis,' in the new famiJY Frankiaceae.

More recently, in a short note, Bumeister and Kausch (1974} claimed

the isolation and in vit~o cultivation of an Aatinomyaes from root nodu~es and

the re-irtfection of llippopha~ plants grown under sterile condition. At th~

time of this writing, their results have not been published.

f) One problem encountered during the isolation trials, was the presence

of a mixed flora inside the. rot nodules. These e~dogenous contam1nants could

not be removed by a surface sterilization of the root nodule. Uemura (1964) , . reported the isolation of about 30 strains of actinomycetes "from Atnus root

nodules. ' Each. strain was grown in pure culture and used in inoculation tests

but without success. In addition to having been time consuming, the nodular o

contaminants May have masked the inability of the cultural medium to sustain

the growth of the true endophyte.

Based on the hypothesis that the endophyte cells are present in

higher 'nuJlber than the endogenous contaminants, Lalonde and Fortin (1972) , used a suspension-dilution technique in order ta get rid of the nodular conta-

minants. In this way they obtained a stock of in vi t1"o-produced nodules

devoid of contaminants. These axenic nodules of AZnuB al'fspa var. mollis will

be shown to permit t,he isolation of a11 actinomycete which showed a morphology 5-

similar to tht of the root nodule endophyte (Section: 5.3.2).

In 1970, duri~g,the purification of the field-collect~d root nodules: l succeeded in isolating an actinomycete showing ~ morphology sin'lilar to the

Toot nodule endophyte (Lalonde, 1971f These re~ults will be presented in section 5.3.1 in order to compare the axenic nodule iS~,laJ:e with the field

nodule isola te , and fina11y, with the root nodule endophyte. It ,-will a180 be

~\

,

/

< > ,

,., v

- 6 -

described in section 5.3.4. how both the field nodule is ~enic

nodule isola te were unable to nodulate aseptic a nitro-,

gen deficient medium. The "possible avirulence 1

been su,gges~ed by Quispel (1960) Jnd Uemura (1964). l

olated endophy~e had

the isolation of the

endophyte, ~n an artificial'growth medium, leads tb a faces the ckfficult problem of relating ~his isolate

(

nodule endophyte without getting the re-infection of

A review of prevjous work suggests that morphological c

be used to correlate a presumptive endophytic ~solate with the true l

root

endophyte. It should be not~d that this crite"rion ls not absolute beca

endophyte of Alnus crispa v,ar. mollis i5 known to be very pleomorphic (

and Fort~n.1973). So, fol'lowing its isolation

medium, an avirul~nt isolate may possibly take a morphology different

in vivo endophyte, thus making its identification uncertain.

one

The actinomycete can be classif ied by combining morphological and

chemical criteria (Williams and Davies,1968; Lechevalier et al,1971). In this

respect, actinomycetal whole-cel1 hydrolysa tes analysed by paper chromatography

for diaminopimelic aeid isomers and for sugars have a recognlzed taxonomie " v~lue (Cummins,1962; Yamag~~hi,1965: Pine and Georg,1965; Lechevalier and

Lechevalier,1968 ,1970; Lechevalier et al,l973). Furthermore, the act,inomycete

may be identified serologically by the specifie fluorescent-antibody technique

(Slack et tj,1961; Lambert et aZ,1967; Blank and Georg,1968; Brock and Georg,

1969; Slack and Gerencsr, 1970; and Holmberg and Forsum, 1973). In add !tion to

a rather similar morphology, the'le chemical and serologieal crit~ria should be D

useful in trying ta correlate avirulent isolate with the true root nodule endo-u

phyte.

Section 5.3.9 will describe how the, two root nodule isolates and the 1r

root nodule endophyte show a similar chemical composition. It will also shown 1

that the immunofluorescence technique permits correlating the field and axenic

noqule is~lates with the root nodule endophyte of A. arispa,va~. mollis. ,"

However, the verification and the localization of the-specifie antigen-antibody

reaction with the immunoferritin method, using th same isolates rabbit anti-

sera, had to be precee4ed by enzymatic degradation of the endophyte capsule. ,

. '

, "

..

" . ... f

r

. Section 4.3 will demonstrat~~at th~ capsule, al~ays surEoundlng the endophyte

cells (section: 3.3), is mostl; compose! of galacturonic acid polyufers .. My ,

investigation of the green silky aIder root nodule ia summar!zed in table I.

It . \:)

\

\

"

\ ~

, r

,-' r

- 8,-' "

TABLE 1 "

Alnus root nodule investigation protocol.

Field nodule

Endophyte

Nodulation

'1

, ) .

Pur',ification __________

Axenic .

nodule

Isolation ~ and

l Field nodule

isolate

Morphology

-Light ~icroscopy

Axenic nodule

isolate

-Electron microscopy

1 ~. Whole-cell Lmmunoreaction

composition -Fluorescein -Ferritin '

o

'1

..

"

l,",:,

e

0

- 9 -

3. ULTRASTRUCTURE OF THE ROOT NODULE ENDOPHYTE.

3.1. INTRODUCTION

, Raving done 1ight microscopy observations of the ALnus criepa var.

mollis root nodule (La1onde and Fortin,1973), l am now attempting, to charac-

terize clear1y the morphology and the cytology of the root nodule end'ophyte .. by means of e1ectron microscopy. Hopefully, this will ptbvide clues for the

isolation of the endophyte.

3.2. ~TERIALS AND METRODS

The whole hast plants were fie1d-collected from the Tadoussac sand

dunes, Quebec. As soon as pO!;lsible, tissues from the tip to the base of the

r?ot nodule wer~ hand eut ~n triangular blocks sma11er th an 1 mm3 and fixed according to the rapid method of Bain and Cove (1971). Following their

glutaraldehyde-osmium fixation in phosphate buffer (0.05 M, pR:7.4), the

nodule blocks were rapid1y dehydrat,ed in ~thanol and infiltrated with pro-

pylene6xide before embedding them in Epon 812 resin. The epoxy resin capsules

were polymerized for 48 h at 60 oe. Ultrathin sections were cut 'with a glass ...

knife, uranyl-1ead stained and examined in a AEl a16B elec~ron microscope

operati~g at 60 kV. "

3.3. RESULTS.

'\

The A. c~ispa var. mollis nitrogen fixing root nodule i8 perennial

and the age of .~l'e noduJ,e tissues increases from the ti; to the b~e. Its . . . , sectioning showed the typica1 pa~ts of a root, i.e. apical meristem, cor~ex

and stele. J~st below the apiieal meristem, which always stayed un-infected,

the h,yphal f~,9f,. the end.ophyte was observed in the host cortical cells

(Fig. 1) .. 1. never observed the endophyte in the stele of the nodule. The hyphal form of the endophyte grew actively in the hast cor,tical cel!, which

, .... staye& a1~ve wi~h its nucleus, ribosomes, mitochondria and 'dictyosomes

(Fig. 4,6,7 and 8). Thete ,was less starch- in the host infect~d cells than . " .

in the un-infected cortical cells. Albeit, the amyloplasts still looked

hea1thy (Fig. 7).

\

- 10 -

The hyphal form of the endophyte was highly septated and could also

be ramified (Pig. 9,10,11 and 12). 1 The septat~on was~continuous with the cel1

wall and the cyto~lasmic membrane/could be seen on each side of the septation

(Fig. 13). Tubulat and lamellar mesosomes were observed in the ,hypba during

its existence in the nodule (Fig. 10,11,14 and 15). In young hyphae, Iamellar 1

~ J mesosomes were often as,.-Sociated with cross septations (Fig. 10 and 11). The

, >

"

...

morphology of the hyphal celi wall was typicai of a gram-positive ai.croorga-

nism and the cytoplasmic m~brane showed a characteristic unit membrane

structure (Fig. 16). The hypha1 cytoplasm was rich in ribosomes and in nu-

clear material but a nuclear membrane was lacking, confirmin. the prokaryotic

nature of the endophyte (Fig. 10,11 and 16). From time to time, ~ome granules

were seen in the cytop1asm of the hypha (Fig. 12 and 16). These granules may

have been sorne kind of reserve material.

, , } --After its penetration il,1to thf!. root via the root hair entrante

(Pommer,1956; Becking,1968; Angulo Carmona,1974], the endophyte, either as a

single hypha or as strands of hyphae, passed t~ugh the host cell wall and

entered the cort~al celI (Fig. 17,20' and 21). The break-down of the host 4t

c~ll wall appearedoto be chemical rather than mechanical. Following these

events, the hypha1 endophyte was surrounded by a unit membrane, probably the

invaginated host plasmalemma. This continuous membrane enve10pe was always ~

present between the endophyte and the hast cytoplasm (Fig. 7,8 and 16). A~ .- /

electron dense capsular material was present between the host membrane'enve-

Lope and the endophyte cell wall (Fig. 12,15 a,nd 16). During the penetration

of the cell wall,' the host cell nucleus migrat.ed towards the infecting hyphae ~

ana increased in size (Fig. 6). In the advanced stage of infection, as obser-

ved in light microscopy studies (Lalonde and Fortin,1973), pthe nucleus was

seen, surrounded by a bundle of hyphae, in the center of the host infected cell

(Fig. 4 and 5).

"After intensive spreading ln the live host cell, the hypha- started

ta produce vesicles near the host cell wall (Fig. 2 and 3). The fiist step - -,

in the vesiculation process was the swelling of the tip of a hypha. In the very young vesicle, the vesicle cytop1asm, whfch was continuous with the

-~ . ~ parental hypha cytoplasm, contaihed a high amount of low electron dense nu-clear material and som~~ranules (Fig. 22). The next step was the formation

, '.

.. S

(

- 1l -

of a septati~n between the parental hypha and the young vesicle (Fig.26).

Figure 26 also shows a large tubular mesosome 8ssociated with the parental

septation . During 'this period of maturation, lamellar mesosomes could also

be seen in the vesicle cytoplasm (Fig.26). The third step, which continues

until the complete maturation of the vesicle, was the oompartmentalization . , of the endophyte cytoplasm by the formation"of septations (Fig:,23,30 and 31).

In each vesicle compartment, nuclear material was distributed (Fig.30,3l and

33). ' The vesicle septation, which i8'a continu~~ion of th~ endophyte cell

wall, grew toward the center

continue tn co~pletion or be ,

septations could be added to

of the vesicle (Fig.3~. This septation cQuld

left un~nished. ~n c~mplete septation, side produce a further compartmentalization of the ,

vesicle (~ig.30). A mesosome was often found associated with the tip of a

growing septation (P-ig.34). Thes~' mesosomeS'" t.ubular or lamellar', were formed , \

by the invagination of the,cytoplasmic' membrane ,(Fig.34 and 35), and were ,

observed from the genesis unt~l the destruction~f the vesicle.

" In the young aseptate vesicle, st~iated bodies cbuld be seen in

addition t04he granules and mesosomes (Fig.28). These striated bod,ies were

still present in the young septate vesicle (Fig.29), but were no observed

in the high1y septate mlJture vesicle.

The vesicle, at aIl ages, was surrounded by an e1ectron dense

capsular mat~ri.al (Fig.27,30,33 and 34). Because the vesicle capsule is

continuous with the capsl,llar material of the parental hypha, its origin must

be identica1 (Fig.24 ,and 25). As in the case of the hyphal form of the endo':...

phyte, the vesicle was a1sif' enve10ped by a continuous unit membrane, p1robably .. the invaginated host.p1smalemma (Fig.30,33 and 35).'-~

Throughout its existence in the'nodule, the. endophyte, as hyphae

and vesicles, was continuously surrounded by a thick (0.1 ~m) electron dense

capsular material (Fig.16 and 30). This capsule was continuous with the hast

cell wall and its electron density was very similar to that of the host cell

wall material (Fig.17-2l). These observ4tions suggest a comparable chernical,

comp~sition. This encapsu1ation of the endophyte wa~ probably the r~sult of

a host cell defenae" reactibn.

,/

\ - 12 -

The crystalline inclusion, shown in f~gures 31 and 32, was only

observed in nodules obtained fram a unique host plant.- This inclusion ~a8 -

observed in the endophyte cytplasm but never in the host cell cytoplasm.

The crystalline pattern suggests a crystalline protein or a cluster of v,iruse.

'~he nodule of the green silky aIder was characterized by a broad tip t _'L-

and a slim base. The slim base was~explained by theJresorption of some host

cells and endophyte forms (Lalonde and Fortin,1973). The fi~st step of the

resorption phenomenon of the endophyte wasl the appearance of dense vesicles

among other normal mature vesicles ~~ig.36). These dense vesicles were " ~

characterized by a very electron pense cytoplasm and a condnsed nucleoid in

each vesicle compartment (Fig. 37). The next step was the p~,ogressive lysis of the endophyte cytoplasm (Fig.3~). As shown by figures 38 and 39, the host

cell cytoplasm also melted away. The complete resorption ph~nomenon gave ri se

to the so-called ghost forms of the endophyte. These ghost forms were charac-

terized by their remaining capsular material and by,some shrunken, and partial-,

ly digested, ce Il wall a~' cytoplasmic membrane (Fig.40). From observations

similar to those of figure 40, l propose the resorption phenomenon might be

regulated! by the neighboring, un-infected host cells.

r.J

tts

"

- 13 -

; .

. ,

,

Fig. 1. Ultra-thin ~t{-on of. nodule tissue, just below

the apical meristem', showing a host cortical cell

/ ' . newly infected.by the hyphal form of the endophyte. CW: host cel! wall, C: host cytoplasm. "Bar is 5,).1111.

Fig. 2. A host cortica'l cell infected by hyphae (h) and by

the Yesi~ular form of the endophyte. The septation 1

phenomenon is taking place in these vesicles. Note

the absence of septation in vesicl~ x, the expanding

one in vesicle y and the completed ones in vesicles z.

CW: host cell wall. Bar f8 5~~m. .i

\ ";.,\. , ,

1

'c

~ . ,

rt

A 1

-14 -~

A host cortical cell containing a high density of , /', 'hyphae (h) .. in !ts center, and septate vesicles (v)

near its ce;n wall (CW). Bar is 5 llm. ,

Fig. 4. A host cell nucleus (N) surrounded by hyphae (h) and

vesicles'(v). CW: ce11 wall, V: vacuole, C: host

cytop1asm. Bar is 5 ~m.

Fig. 5. Detail of figure 4, showing two hyphae (h) surrounded "!:~;/';~ .... ,. t~; bv the nucleoplasm of a host cell nucleus. Note the

~'__ nuclear membrane (NM) and the plsma1erma (P) enve-loping the hyphae. Bar is l llm.

o

- 15 -

Fig. 6. Another view of a host cell nucleus (N) surrounded

by hyphae (h) and veslcles (v). Note that this

nucleus i9 adjacent to the site 'of penetration of

the host cell wall (CW) by a hypha. V: vacuole, l ..

NU: nucleolus. Bar is 1 ~m.

Fig. 7. Along side the hyphae (h)" a zone of host cytoplasm

\ shows mitochondrioh (M) and a small starch grain (SG)

contained in an amyloplast (A). Note the plasmalemma

(P~ enveloping the endophyte hyphae. Bar ls 1 ~m.

Fig. 8. Nearby the hyphae a zone of host cytoplasm shows a

mitochondria (M), ribosomes (R) and a dictyosome (0).

Note the plasmalemma (P) between the host cytoplasm

and the hyphae (h). Bar is 0.5 llm.

"

1

r

.'.

If !

.,

>

1 1 ~ ...

r 1 '.

Fig. 9,-

- 16 :""

." Jt"> ;

and transversal section~ of hyphae (h) 1

infected host cortical cell. Bar is 1 ,~m. )

1 f

Fig. 10. Detail of figure 9, showing the ramification of ,the

hvpha. Mesosomes (m) can be seen associated with

septations (s). n: nuclear material. Bar ia 0.5 ~m.

Fig. 11. Another view of mesosomes (m) in young hyphae. Note

the septations in the lower hrpha. n: nucle~ma-terial. Bar ia 0.5 ~m. JI!

\ :r

, _J

".

'- , 1 :

D

..

",

1 t,

- 17

o

Fig. 12. Hypha showing septations.(s). An electron dense

capsular materia.l Cc) 1(i seen 8urrounding the hyphae .. ~ p

. Ch) and the partially seen vesicies (v). g: granule. , ,

Bar is 0.5 llm

,

. Fig. 13. Detail of a hyphal septation. Note the contin~ity of

Fig. 14.

Fig. 15.

the cell wall (cw) and of the cytoplasmic membran~ "

(cm), forming th septation. A capsular material (c)

ls seen aIl around the hypha. Bar is 0.5 llm,

Transversal section of hyphae showing a tublar

mesosome (tm) P: host plasmalemma. Bar is 0.5 llm. "

.dl il

, '

0

Transversal section of 'a hypha showing another type; "

of tubular mesosom~ (tm). P: host plasmalemma, d ~

c: capsu~ar material, cw: endophyte cell w~ll,

cm; endophyte ce Il membrane. Bar is 0.1 llm. ,ff'

,t, ,r

"

P;

~>,. \\

"

IJ

Fig. 16.

1

'1

- 18 --

, ),

Longitud inaL sec t ion of a young

granules (g) in its cytoplasm.

".

~ hypha showing

The'hypha is

, surrounded by a capsular matrial (c) and by

the host plasmalemma (P). cw: endophyte cell

wall, cm: endophyte celi membrane, n: nuclear

materiai. Bar is 0.1 ~ .

./

, ,

\ ~

---, --~--~-~-----~--

(

r

J

.'

, ,

!

1'9

Fig'. :7-21. Penetration of hyphae through the host ce11 wali~~~~ e (

Note in these electron micrographs the continutty

, (un1abe11ed arrows) of the capsu1ar material (c)

with the host ce1l wall (CW).' Note also the s~i-c

1arity in lectron density between the capsule and

the cell wall materials. V: vacuole, h: hypha,

v: vesicle.

Fig. 18-19. Details of figure 17. o

Fig. 17. Bar i8 1 lJm.

Fig. 18-19. Bars are G.5 lJm.

Fig.-ZO-21. Bars are 1 lJm.

f

, ,

, 1

1

(

, \,

\

..

--------4-_____ _

-----

"

" ,

---------

\

,.,.

\

20

Fig. 22-26. These electron mic~o8raphs were selected to

show the development of a vesicle by the

swelling of the tip of a parental hypha.

Fig. 22.

Fig. 23.

Fig. 2'4.

-........ The tip of a parental hypha (ph) beginning

to swell. Note the absence _of septation, . .

the high content of nulear materiel (~), f

aAd the" presence of granules (g). "Bar is'

1 \.lm.

J

A young vesicle adjacent ta the hast ce II

wall (CW). Meso,somes (m) can be seen near ~

the newly formed vesicular sept~tions (s).

The nuclear material (n) ls now dLstributed

in the vesicle compartments. Note the dif-

flculty in sectioning the point of attach-

ment of the v~sicle with its parental hypha

(ph). Bar ls 1 \.lm.

" Ultra-thin section showlng the continulty

of the capsular material (c) from the paren-

tal hypha (ph) to the young veslcle (v). 1

Bar ls 0.5 \.lm.

!

L_ I ---- ---Fig:-- 25: --'- '-uustained vesicle (v) observed on,,.-'--formvar

coated gr'ld.' Note the capsular materl~l (c') surrounding the parental hypha (ph). ~ar ls

l \.lm.

"

"

/

..

... ' . '.

. ~ .. '

.

... '

1

.' , ~.,..

'",

-

,: /

1

1

1

- 21 - 1

Fig. 26 .J.n aseptate young vesicle ,produced by the terminal

swelling of its parental hypha. Note the parental

septation (ps) at the origin of the vesicle. Ad-

jacent to ~his hyphal septation, tubular aesosome

(tm),can be seen. Lamellar mesosomes (lm) are shown

in the vesicle cytoplasm. Note the hi~h number of

granules (g) and the abundance of nuclear material

(n) in this young vesicle. Bar 15 0.5 ~m.

Fig. 27. A rare "twin" vesicles showing a commune capsular , . . ~w material (c,arrow). Note the absence of the host

membrane envelope betwen these tWo vesicles (v).

Bar is 1 ~m.

.:

" "

.-

.'

e ~o~ i

., @ \

, " n r, 1

'" . ,

,b r ,. ~ 1-

( ~,~

r' .

41 ...

-(~;~ .. ., . . .'. f '. ' r. . ( -:~ " ,

l'

. ,

f

J

- 22 -

Fig. 28. Two aseptate young vesicles (v) showing striated

bodies (sb). Note the high content of nuclear

material (n) in_the vesicule cytoplasm. h: hypha.

Bar i8 0.5 ~m.

Fig. 29. Aseptate young vesicle showing striated bodies

longitudinal~y (sbl) and transversaIIy (sb2)

sectionned. s: septation. Bar is 0.5 ~m.

..

r

,

) .

23 -

Fig. 30. Adjacent to the host cell wall (CW) , a young vesi~le with

a complete septation (a)/shows a side septation {si;) in

formation. Granules (g) can be seen in the vesicle cy- " ...

toplasm. Ribosomes (r), mesosomes (m) and nuclear mate- ~

rial (n) are seen in each vesicle compartment. The

veslcle ls enveloped by t~e host plasma unit membrane (P). N~te the void area .(va) between the endophyte cell wall (cw) and the capsula~ material (c). V~ vacuole, . T: tonoplast, m: endophyte cytoplasmic membrane. Bar is 0.1 ~m.

~~---o---

,

, ,

, , , , ,

, y , ,

-~--- -. ~~~~-

< .

"

"

-,---.---------.---~--;----------

; )

" .,.'

" '! 'ia '-"" ...... . . ). .' ,,~ ! "

V ~

~ ..., -. - ,. , .. ______ c ____ _

~~ _____ LJ __

... '

1

. -'

.'

1 c> .

- 24 -

1

, ....,

f Fig. 31. A highly septated maturing vesicle, showing the formation

of side septations (ss')' from complete? septa (s). Note

the crysta~line inclusion (ci) in the cytoplasm. The

nuCTar material ~J1S--easny recoginzable ~fs~ea. of----low electron density. cw: endophyte cell wall. Bar

19 0.5 jJm. t l

,.il

Fig. 32. High magnificatioO of a rystalline inclusion (ci) seen , of

in a vesicle similar to the one shown in figu~~ 3l.

~ Note the well defined pattern1.)of black dots in arrays. Bar is 0.01 lJm .

v

"

"

/ .. 1-' ." "

" ..

\

.. "

"

, '1.

.,

...

;;

.' -, \ JI~ .. _,

\, ~ ,

"

- 25 1 o

(

'\ . Fig. ?3. Detail of a mature~vesicle showing the continuity of the

septation (s) ~lth the cell wall (cw). Compare the

structure of ~he lamellar mesosome (lm) with the weIl

resolved cytoplasmic unit membrane (cm). A nuclear ,

membrane is lacking but the nuclear material (n) ls seen as fine threads. Ribosomes (r) are abundant ,,in the en-dophyte cy~oplasm. Note the void area (va) between the

endop~yte cell wall (cw) and the capsular material (c)

surrounded by the host plasmalemma (P). V:" vacuole,

T: tonoplast. Bar i8 0.1 ~m.

/

o

(

1

--- --" ------ ------~ -----;:;-- - - ---

(

-". .. t

r'

o t~HJ' "',:-, 1 ......

,1

r, r--------

"

- 26 -, 1)

Fig. 34. This electron micrograph shows a lamellar mesosome (lm)

being producd by the invagination of the cytoplasmic

membrane (cm). s: septation, va: void area. Bar i8

0.1 IJm.

. )

o

Fig. 35. This ul ra-thin section il1ustrates a tubular mesosome .. (tm) s i11 attached to the tip of a vesicle septation

(8). ote the plasmalemma envelope (P) surrounding

each v sicle (v). va: .void-,area, c: capsule. Bar 18

O.l.~m .

.....

-------~--;--- ----

, "

..

---~------

;

:~ l'

o

..

. -.

------------------------------

Fig. 36.

Fig. 37.

- 27 -

,

These electron micrographs show the sequence of

events during the resorption phe~omenon of the

etldophyte. 'V-

An infected ho st cortical cell in the basal part

of the nodule. Note the electron dense vesicles

(dv) which can be seen with other normal ves~les

(v) and hyphae (h). CW: host cell wall, V: vacuole.

Bar 18 5 \,lm.

\ .. -...

In this septate dense vesicle, note the condensed

nucleoids. (n) and the presence of granules (g).

c: capsular material. Bat i8 0.5 \.lm

1

\

" .

_------------------- ----_._----------------------

"',

- \

p

" r

lI;.

1

..-z8------- ------.---

Fig. 38. This micrograph demonstrates that the formation of dense

vesicles (dv) i9 foll~ed by the resorption of the

endophyte cytoplasm. The partially resorbed vesicles \

(rv) still contai~dense masses of cytoplasm, but the

complete resorption gives ri se to the ghost hyphae (gh)

and ghost vesicles (gv). NOte that the host ,~ell <

cytoplasm also melts away. CW: hast cell wall~ Bar is '" , 1 lJm. 1

~, , ; .. ~

Fig. 39. This microgra~h demonstrates that the endophyte ghost

forms are nat the result of a fixation or embedment

artefact. Note that in the adjacent non-infected host

cortical cell, the cytoplasm contains a weIl preserved

nucleus (N) and starch grains (SG). CW: host cell wall,

V: vacuole, TC: tannin cell. Bar is 5 ~m. '

'.

Fig. 40. High magnification of th;'ghost hyphal (gh) and ghost

vesicular (gv) forms of the endophyte. Note that only

the endophyte capsular material (c) and some'shrunkeJ deb~is - - ~-- ----- - -- -- - -- --- --- ---- ---(sd) rematn visible. Bar i5 1 ~~ .

. ..

f

'.

1-------- ____ _

1

r s

..

.,.

29

3.4. DISCUSSION

~ The penetration of the" host cortical cell wall by the endophyte was

also r~ported by Silver (1964), Becking et al (1964) and Ga~dner (1965).

Furman (1959), in his light microscopy study of Ceanothus root nodule, suggest~d

that the plasmodesma may have provided the passage route for the endophyte 9 \

from one host cell to another. However, the 'pore si~e'of the plasmodesma is

too small to permit a direct penetration of the host cell wall by a hypha.

l agree with Silver (1964), that the penetration is chemica). rather than

mec hanical .

... ,,,'" f. '"

Figures 22,23 and 26 demonst rate the fo.rmation of the ves~il.-~y

the terminal swelling of a parent b-ypha. These electron micrographs confirtned

our light microscopy observation of the same event (see Fig.S in Lalonde and

Fortin,1973). This mechanism of formation was sug~~sted for the A. gZutinosa

root nodule endophyte vesicle (Becking et aZ,1964) and demonstrated with

HippophaM rhamnoides root nodule endophyte (Gardner and Gatner,1973),

Mesosomes refer to sac-11ke invagipations of the cytoplasmic mem-

brane, together with the tubular, vesicular and lamellar membranous contents

of these sacs (Rcavely and Burge,1972). Mesosomes have been observed in gram-

pos~tive and gram-negative bacteria (Fitz-James ,1960; Pate and Ordal, 1967;

Rucinsky and Cota-Rob1es,l974; Williams et al,l973) and in actinomycetes

(Chen,1964; Duda and Slack,1972; Overman and Pine,1965). Mesosome-like

stru~tures, as. p1asmal~mmosome or 110nion body", have also been observed in the

different non-Ieguminous root nodule endophytes so far studied (Becking et al,

1964; Gardner,196S; Gatner and ~ardner:1970); but their role is not understood,

- lrnough 1t must be recugnized tnat-th~y are related ~o septation formation in

hyphae and in vesicles' (Fig~O, Il,26 and 34).

It.. was shown j

t~at granular inclusions could be seen in most endophy-,.

te forms. But because only a very small.quantity of this material has been , seen in an actively maturing vesicle, l am lnclined to believe that the granul~

is a reserve compound. Furthermore, thefo~ of, the inclusion and its electron 1

density suggest a stmilarity with the non-lipid po+ar body of rhizobial bacte-

roids of Lotus noctules (Craig et aZ,1~73). . \ /

- 30 -

The crystalline inclusion seen in the endophyte vesicle seemed to

be a crystallized protein (Fig. 32). ~ Its structure ~as very similar to the

crystalline_inclusion induced by tobacco etch virus in its hO$t plant (Mcdo-

nald and Hiebert,1974). The presence of poorly lytie phage in actinomycete

has been reported (Higgins and Lechevalier,1969). Therefore it ls tempting

to specula te that the crystalline inclusion probably resulted from phage in-

fection. ~oreover, this endophyte inclusion has not been observed in other . non-Ieguminpus root nodule endophytes.

The characteristic capsular material that always surrounds the ~

endophyte of A. opispa var. mottis root nodule V3S also'shown to encapsulate

the root nodule endophyte of A. gtutinosa (Becking et at,1964; Gardner,1965),

/ Mypica oerifepa (Silver,1964), HippophaH phamnoides (Gatner and Gardner,1970;

Gardner and Gatner,1973;~Baumeister a~d Kausch,1974). This capsular material

has been seen around aIL stages of the endophytes found within the non-legu-

minous root nodules so far studied. From staining and degrada~ion studies

l have elucidated the granular and fibril1ar pectic nature of the endophyte

encapsulation material and its probable biogenesis by the plant host cell

(See section: 4.3).

At the time of the penetration of the host cell wall, the hyphal

endophyte was surrounded by the invaginated hast plasrnalemma and seemed to be .. enveloped in it during its existence in the nodule. Because the host cell

(.

wall was very dense compared to the host cytoplasm, it was very difficult ta

obtain a uniform ultra-thin section 'of the nodule tissue. Thus, l never fully

resolved, on one ultra-thin section, an invaginated plasma membrane surrounding

the spreading hypha. One may suppose that the membrane envelope originated

from the endoplasmic reticulum or de novo. For the time being, these sugges-

tions are as good as my"plasmalemma-origin"assumption. One possible way to

close the debate would be the loca1ization of a marker enzyme, such as the

adenyl cyclase plasma membrane-bounded enzyme, to study the relationship

between the endophyte mel'\1brane envelo'pe and the host plasmale1lDDa (Tu,1974). ,

Gardner (1965), using A. gZutino8a root nodule tissues fixed in 1<

buffered osmium fixative, note~the gccur~~ce of irregularl~ shaped,vesicles

with very dense contents. But she did not correlate them with a resorption

/

\ f

~ 31 -

c

event, as 1 do in the present study. However, Becking et al (19~4) did not report dense vesic1es in their study of A. glutino8a ro6t nodules. It shou1d

be pointed out that, because the ~ense vesicle forro seemed to be very ephemer~

and was on1y present at the base of the nodule, the chances of seeing it ar~

slight.

,The endophyte ghost forms, resulting from the comp1eted resorption

phenomenon, were a1so observed in light microscopy study of A. cpispa var. -,

molli8 root nodule endophyte (Lalonde and Fortin,1973). This phenomnon of ,

re~orptio~ of the endophyte in old basal tissues of the root nodule was also , A

reported in A. glutinosa (Becking et al,1964). This resorption phenomenon may , }

reflect a disequilibrium in the host-endophyte symbiotic interaction with the,

10ss of the nitrogen-fixation potentiality.

oBacteria-1ike cells, prev;ously observed in the light ~icroscopy

study of A. cpi8pa var. molli8 (Lalonde and Fortin,1973), were not seen in 'the

present work. The~r presumptive absence in the numerous nodule tissues stu-

died may reflect a rarity of this endophyte form in the green silky aIder root

nodule. 1

o

r

- 32 -

4. ULTRASTRUCTURE, COMPSITION AND BIOGENESI'S OF THE ENDOPHYTE CAPSULE.

4.1. INTRODUCTION.

r So far, the encapsulation of the endophyte of non-leguminous root

nodules has been obeerved in aIl e~ectron microscopy studies (Silver,1964; ~

Becking et aL,1964; Gardner,1965; Gatner and Gardner,1970; Gardner and Gatner,

1973). It has been suggested that the capsular materia1 represents a poly-

saccharide (Becking et al,1964).

"

l think that this encapsulation phenomenon, which f0110ws the pene-

tration of the endo~hyte in the host ~ortical cells,- fuight represent an

important feature of the actinom~cete-angiospermous nitrogen-fixing root nodule 1

symbiosis. So l looked for the characteristics of 'the capsular material oy the -l>

use of staining techniques and enzymatic treatments, fo110wed by light and

electron microscopy observations. In addition l have investigated the biogene-

sis of this endophyte capsule.

4.2. HATERIALS AND METHODS.

4.2.1. Band eut sections of root nodules.

The fresh or the rehydrated pre-fixed root nodules [formaldehyde or

FAA fixed (Jjhansen,1940)] were hand eut ,with a single edge blade. These ilec-c ,

tions were than pod1ed in Petri dishes co~taining a saline solution (0:6% NaCl)

and used, as soon as possible, in the different staining tests. ~

4.2.2. ranyl-lead stained nodules sectidhs. J

Root'n6dulei blocks (!mm3) were glutaraldehyde-osmium fixed according

to the rapid rnethod of Bain and Cove (1971). Following their fixation, tle

tissues were rapidly dehydrated in ethanol and infiltrated with propy1ete oxide

before embedding in Epon 812. The epoXy resin capsules were polymerised for

48 h at 60C. Ultra-thin sections were eut with a glass knife, urany1-lead

stained. and examined in a AEl E116B electron microscope operating at 60 kV.

- "

7

\

- 33 -

4.2.3. Purified endophyte~suspension

,~ J4 . Fresh field-collected root nodules of Alnus crispa var. mollis were

surface sterilized by three minutes of'agitation in 10q ml of 5.25% NaOel

solution. The nodules (10 gr) were washed 5 times in ,sFerilized distilled

water. These nodules were c'rushed for 3 minutes in saline "Solution (0.6% N1,Cl)

with a Virtis homogeni-ser""'working at high speed in arder ta release the endo-

phyte from the host cortical cell. The resulting nod~e-suspension was added r- ,

to a 250 ml graduated cylinder and topped with saline solution. Following~the

sedimentation of mast hast tell wall debris, the supernatant containing the

endophyte vesicles with theit so characteristic small parental hypha, still

attached tJ them, was resuspended and sedimented two more times. The endo-, , phyte-suspension was then centrifuged at high speed in an International Clini- ,

cal Centrifuge, resuspended in sal!ne solution and recentrifuged until the

complete elarifiaation of the supernatant. The purit~ of the endophyte-suspen-

sion was ehecked in phase ~ontrast mieroscopy and the suspension had to show

a content of over 70% endophyte cells .

. 4.2.4. Polysaccharide staining test~~

4.2.4.1. Alcian blue.

4.2.4.1.1. Light mieroseopy.

I~nd cut sections of root nodules were stalned in 1% Alcian Blue 8GX .. f ~ ,

(Allied Chemieal Corp., N.Y.) for 10 minutes.L The dye was di~solved in 0.5N HCl

to provide a solution of a,pproximately pH Q.5 fnd in 3% aeetie acid for a.plI 2.5

solution (Lev and Spicer,1964). 1 t,f ,1

4.2.4.1.2. Electron mieroscopy.

Blacks (1 mm3 ) of nodule tiss'ues were staJned in 1% Alcian Blue 8GX

in 0.6'% saline solution, fixed 15 minutes in 6% glutaraldehyde, washed and

post-fixed in 0.5% Os04' After washing with distilled water, the blacks were j,

ethanol dehyd~ated, propyleneoxide'infiltrated and Epon 812 embedded . ... ~r'" 1

- 34 -

4.2.4.2. Calcofluor staining. ':;-

4.2.4.2.1. Smears.

Heat fixed smears of purified endophyte-su~pension were stained for

5 minutes in a 1% water solution of Calcofluor '~lite M2R New (The fluorochrome ,

was kindIy suppl{ed by ~r. J.M. Duckett, sales manager of Cyanamid of Canada

Ltd., ~ontreal, Quebec.). After 3 successive washings in~dl*tilled water, the

glass slides were then observed in incident-light fluorescence microscopy .

4.2.4.2.2. Nodule sections.

Fresh nodule tis~ues blocks (1 mm3) were stained for 45 minutes in an 1% solution of ca1cof1uor, fixed 15 minutes in 6% glutaraldehyde, washed

5 times in phosphate buffer (0.05M, pH 7.4), fixed in g.5% Os04' washed, dehy-

drated and embedded in Epon 812. Thick sections (10-100 ~) Mere then obser-

ved in incident-light fluorescence micropcopy. " ......... -

4.2.4.3. PAS reaction.

".

PAS (Periodic Acid-Schiff's) reaction (Jensen,l962), was used on

fresh hand-cut sections and on Epon sections of 5% formaldehYrle fixed root

nodules in order to detect total carbohydrates of i~soluble polysact~arides. , .~

4.2.4.4. IKI-H2S04 reacti~n.

Cellulose t~ fresh, hand-cut se~tions of root nodules was tested for, using the IKI-1I2S04 method of Johansen (1940).

"

4.2.4.5. Aniline blue.

Callose was t~sted for by staining Uand-cut ~ections of root nodules

with Aniline Blue (Allied Chemical Corp., N.Y.) in water and observing"them by

ultraviolet light, as describ.;,d by Currier (1957).

'-

- 35 -

4.2.4.6. Ruthenium red.

Pectin-like substances of hand-cut sections of root nodules were \ .

observed by staining with Ruthenium Red (BDR Chemical) according ta' the method

of Johansen (1940).

~ 4.2.4.7. Ruthenium Red - Os04'

Acid polysaccharides were tested for using Luft' s rutl)enium ied ,-

Ps04

method (Luft,l97l). A saline solution (0.6%) wai used'instead of a

,cacodylate buffer. After the staining and fixation processes, the endophyte-- \

su~pension, enzyme treated or untreated, w~s centrifuged, agar enrobed, ethanol

dehydrated, propyleoxtde infi1trate~ and Epon embedded-. Ultra-thin sections

_were~served without further staining.

4.2.4.8. Rydroxy1mine - ferri-c ch10ride reaction.

Pectin substances of hand-cut sections of root nodu1s were specifi-

cally localized by th~/hydroxy1amine-ferric ch10ride reaction of HcCready and

Reeve (1955). When n~eded~ the over-night esterification of the endophyte capsu1ar material was accomp1ished by placing the hand-cut sections (fresh,

rehydrated or pre-fixed) in solution of absolute methanol containing 0.5 N

hydroch1oric acid. , '.

4.2.5. Enzymatic degradatioR of the endophyte capsule.

A 0.5 cc pe.11et of a pur if ied endophyte-suspension was resuspended fv1 in 10 ml of phosphate buffer (0.05 m, pH 6.0). Ten mg of each antibiotic

c,

Penicillin G Potassium (Ayerst Lab." Montreal), Acti-dione (Upjohn Co., l1ich),

and Chloromycetin (Parke-Davis Co., Ltd., Ont.) was added-with 50 mg of the

enzyme to be used in the degradation test (Purified Pectinase from Aspergillus

,niger, No-P-4625; practical grade Cellulase type l from Aspergillus niger, ,

No. C-7377; crude Remice11ulase grade'll from RhizopU8 mo1d, No. H-2125; ,

(Sigma Chemica1 Co., St-Louis. U.S;A..

1 'c

-

J

\

_ e.

- 36 -

The glass tube, eontaining the sample, was-clG-sed wi th a rubber--

serum stopper and ineubated forJ24 h at 28 DC. At different intervals, samples

were eentrifuged and washed 3 times. The resulting pellets were used in the

different staining tests. When the supernatant was assayed by thin layer

ehromatography for the capsule degr~dation produets, the TLC plates (Siliea /'

Gel,lB2, 2Ox50 cm, Baker-Flex) were spotted (5-100 pl) with the supernatant

and control sugars (Sigma Chemicals Co., St-Louis, U.S.A.). A h-butanol-

acetie aeid-water (50,25,25) so~vent system was used for th detection of-

galacturonic aco' and~lucur6nic acid, and a n-bptanol-pyridine-toluene-water . ,~

(100,60,80,60) solvent system for the neutral sugars.~ \ '\"\"\"\,

A" -#-9

"

~

- 37 -; 1

Furthe~re: the immunolabelling of the e~OPhY4- capsule vas done with the antiglobulin, method by using rabbit antisera, proruced by intravenous

injections with washed formolized whole cells of a purifie~ ~ndophyte-suspen-~ , sion' (section 4.2.3 .

~ . 4.2.7. Solubilization of the endophyte capsule.

A ~resh endophyte-suspension was shaken, centrifuged and resuspended

10 times in disti1led water. Fo110wing this treatment, the endophyte-suspen-.r \ sion was formaldehyde-fixed and the endophyte capsu1ar material was specifical-

1y 'labelled with an immuno-ferritin complex ~y the in!;!irect immuno technique.

The thickness of this treated, ferritin-labelled, endophyte capsular material

was then compared ta that of previou~untreated endophyte capsule. The purpose , of this test was ta check the water solu't5ility of the endophyte capsule."

4. 3 : RESULTS.

4.3.1. Electron microscopy of uranyl-Iead stained nodule sections.

, The primary infection of the A Znus IJl'ispa var. mollis root nodule

host cell is done by the hyphal form of the endophyte. Figure 41 shows that

th hyphae were able to pasa through the host cell wall. Figures 42 and 43,

moreaver sugges': that the endophyte hyphae \lere able ta degrade the host cell ,

wall components and particularly the pectinic Middle lamella; this degradation /

/ is probably enzymatic. Following their brief growth in the host cell wall,

_ ~ __ ~~ the hY~h~=-emerged into the adjacent hos't cell and spread from the site, of l -----tnfe-cn:.mr i:nto the cortical celle ~oon --cn~nfecting- hyphae -lett 1:he~

, cell wall, they were seen to be encapsulated by a thick electron dense mater1al,

the so-called endophyte capsule. Figur.e 43 suggests that the endophyte capsule, v -.

whic~ is continuaus with the host ~ell wall, might be the prolongation of the middle.lamella. The same figure also demonstrate that the primary wall extend-

et in a very short sheath forming a collar-like structure around the infaction

hyphae.

" Following the penetration of the hOpt cell, the endophyte hypha was 1

enveloped in the.invaginated plasmalemma. This 'hast membrane envelope always

- 38 -

isolated the hYPhal or v~sicular endophyte from the host cell cytoplas~. When

&J the hyph~e emerged from the host cell wall, this membra?-e envelope was often

\ masked due to the accumulation of an electron opaque substance covering the

hyphl surface (Fig. 43). No explanation can be offered as to the nature and

origin of this subst~nce. ,

"-' ........... .............. Theae electron microscopy observations of the glutaraldehyde-osmium lf ........ ~ .. ,,'>'t.~

, . fixed and uranyl-Iead ~tained ultra-thin sections of AZnU8,root,nodules, sug-gest t~at the endophyte capsule, which is continuous and shows an electron

density similar to the host cell wall, might be analogous in chemical composi-

tion.

4.3.2. Electron microscopy of alcian blue stained nodule tissues.

The host cell wall and' the encapsulat!on material of the endophyte

were seen as fibrillar materials ,after their glutaraldehyde-alcian blue-osmium , fixatiop (Fig. 45 and 47). However the pH of the alcian blue - glutarald~hyde

" fixative used 'in this investigation (pH 6.0), was too high to stain polysaccha-

ride specifically. The fibrillar material that surrounded the hypha, also

encapsulate4 the vesicular form of the endophyte (Fig. 44,45 and 46). The

alcian blue staining method merely corroborated the continuity of the fibrillar

capsule with the host cell wall.

\ '?t "

4.3.3. Fluorescence microscopy of calcofluor stained nodule ti~sues.

\ Figure 48 shows the endophyte capsule and. th host cell wall reac-

ting s~~ilarly~i~~,the polysacCha~ide(Calcofluor White M2R New ~tain~ Fur-the~more, a comparison o~ ~igure 48 with figure 49 demonstrates tlr specificity

of the fluorochrotne in staining on1y the. host cel1 wal.l and the endophyte

capsule. The host cytoplasm and the starch grains remained unstained. In

,addition, figures 50 and 51 prove that a calcofluor positive capsular material , surrounded the hyphae, the vesicles and the ghost forms of the endophyte. The

reactivity of the endophyte capsule with the ,calcofluor brightening agent de-

monstrated the polysaccharidic nature of the capsylar matrnal (Nagata and" t

Takebe,1970) . r;

... ~ 1)

,G)

- 39 -

4.3.4. Periodic acid Schiff's (PAS) reaction on nOdule sections.

1

Table II shows that the PAS reaction corroborated the polysaccharidic

nature of the endophyte capsule as suggested

addition, SchHf' s reagen.t ,is known to react

ter calcofluor stain. In

without prior oxidation (Jensen,l962); therefor II also demonstrates

th~\ the endophyte capsule was not a lignin material. Figure 52 illustrates'

the fact that the PAS reaction took place in the capsular material of the endo-. phyte but not in its cytoplasm. The same light micrograph also demonstrates

that the polysaccharidic mat'rial was present around both the hyphal and the

vesicular forms of the endophyte.

4.3.5. Cellulose s~aining 6f nodule sections.

The classic cellulose-IKI-H2S04 method was ~ried on fresh sections

of root nodules. The host cell wall was stained a dark blue but the endophyte

capsule remained clear. According to this reaction; the capsular metarial of

the endon~yte i5 not a cellulose-like material.

4.3.6. Callose staining of nodule sections.

After the aniline blue staining of fresh root nodule sections, the Il ,

incident-light.fluorescence microscopy observations of these sections s~wed no

fluoresc

reaction

4.3.7.

endophyte capsule or of the host cell wall. Since the

blue with callose is specifie (Currier,1957), these obser-

that the endophyte capsule is not velated to a callose p

1

blue staining of nodule sections. " .. ,

Alcian blue hs commonlY,peen used in light microscopy as a stain ""

specific for acidic polysaccharide at low pH (Lev and Spicer,1964). At pH 2.5, ~

the cationic alcian blue dye forms complexes with carboxyl groups and/or sulfa-

te grQUps. At pH 0.5 the carbvxyl groups of the acidic polysaccharide do not dirsociate so that alcian blue complexed exclusively wfth sulfate groups.

Talhe III shows that at pH 0.5 ,the endophyte capsule did not react witq ~he

, 1-- - --

m $

.. 1 ....

- 40 -

TABJ.E II J

o

~AS feaction of A. c:r,oispa var. moHis nodule sections. ~ ~ ~

1.

Schiff 1 S reagent

PAS react ion

Endophyte

capsule

+

Host cell

wall

+

Host cel!

cytoplasm

Symbols-,----aemained--C.lear 1 :became red. a The root nodule material was either fresh or fixec:r:-rrfixd, the -fiMute----~

section was rehydrated ~rior to the periodic acid oxidation

. , '1 ",

..

\ ' '-1,. "

o

s f r :J-'

- 41 -

TABLE III

Alc1an blue stain1ng of A. arispa var. moZZis Toot nod~le sections. a

1

Endophyte

capsule

Host ce1h

wall

%

pH 0.5

pH 2.5 +

( Symbuls-,~ -a:el'llained clear; +:bec8JlL~ blu~. aTested on fresh and fixed root nodul~ sections.

..

+

"

Host cell

cytoplasm

"

\

\ 0

'\"

- 42 -

aleian blue dye, thus demonstrating the absence of sulphate group in the

capsular material. Therefore the positive reaction shown at pH 2.5 demonstra-

tes the acidic nature of the endophyte capsule. (

4.3.8. Ruthenium red staining of nodule sections.

The classic procedure for localizing J>ectic substances is the stain-

ing.with ruthenium red (Jensen~1962). The ruthenium red stained~oot nodule

sections gave a positive reaction in the middle lamella of the host cell wall

and in the endophyte capsule. These observations strongly suggest that the

capsular material is a pectic substance. However the staining reaction of

acidic polysaccharide with the ruthenium red dye, demonstrat,ed that the classic-t.

al reaction of ruthenium red with pecti~ substances is typicai rather than .

specifie (Luft,1971). The reactiviS1 of the endophyte capsule with the ruthe-

nium red dye corroborated the acidic nature of the ~olysaccharide already

suggested by the alcian blue reaction. For this reason the cObfirmation of

the pectic nature of the capsular material must b.e demonstrated b'y o.ther stain-

ing techniques.

4.3.9. EM observations of RR-Os04 stained en~ophyte-suspension.

In the afore mentioned ligh~ microscopy observations of the rut he-

nium red-stained root nodule sections, the staining reaction was used to demons-

trate the acidic nature of the capsular polysaccharide. For electron microscopy,

th~ reaction of ruthenium red with osmium tetroxide may be used to ampIify the

electron density of the stained .acidic polysaccharide material (L\Ift, 1971) .

This' property of RR-Os04

was used to characterize the ultrastructure of the

,,~phyte capsule.

Figure 54 shows the strong contrast of the endopJlyt~ capsule after , it was stained with the. ~-OS04 mixture. 'In figure 55, the stainin~.dembnstra-

. '" t"es that t'he endophyte capsule is formed of granular and fibrillar materials. In addition, Hogure S~ shows that the RR-OsO 4 stain permits the differentiati?n

of the endophyte capsu~e into two-layers: an inner, thin, and dense lin~, adja-

cent to the endophyte celi wall, and an outer, granular and fibrillar layer. '~ .

Since these two regions'of the capsule reacted strongly with the RR~Os04 stain,

, 0

\

,

l conclude that both cemponents ate c-"~o~ acidic Knowing that they exist, the two layers of the endophyte

polysaccharide.

capsule can also be

Iocated on a glutaraIdehyde-Os04

fixed and uranyl-lead ~tained ultra-thiu ;

section of a root nodule (Fig.65) or on a glutaraIdehyde-Os04

fixed ultr~,thin

section of an endophyte-susp~~sion (Fig.67).

4.3.10. Hydroxylamine-ferric ch10ride reaction of~nodule sections. "

The alcian bIue staining tests have demonstrated that the capsular

material were an acidic polysaccharide. This result was further corroborated ./

by the ruthenium red stain which a1so suggested that the a~idic polysaccharide

might be a pectic substance. The highly specifie pectin 10calization procedure ~

of '-1cCready and Reeve (1955) might determine t'~e enigmatic nature of the acidic

polysacch~ride.

The McCready and Reeve's method is based on the reaction of alkaline

hydroxylamine hydrochloride with the methyl esters of pectins to produce pectin

hydroxamic acid, which, in turn, produces brown complexes with ferric ions. .. --The coloration in the untreated tissues depends on the amount of esterified

pectins and on the degree of esterification. This Iast point is demonstrated

in table IV.

Tahl e V demonstrates the fact that the 'kCready and Reeve' s pectin~

teSl!: permitt;!.d the identification of the endophyte capsular material as a de-'..(

esterified ~ecti~ substance. The pectic substance is illustrated in fig~e 53

by t~e Rtrong staini~~ of the host infected cells, containing the encapsulated

endophyt~>cel1s. This hand-cut section was methyl esterified bdore the

hydroxylamine-ferric chloride reaction . .. ~ . ,

4.3.11: Insolubilitv of the endophyte capsule.

Fig~re 58 illusEtates the aspeet of the ferritin-labelled hyphal

capsule after the water solubilisation test. Furthermore, the comparison of -

the in vitro hyphal capsule shawn in figure 58, with the in situ hyphal

capsule of f igur~ 6'5, demonstrates that the water treatment did not decrease

the thickne~ of the endophyte capsule. In later observations, the vesicular

"

- 44 -

TABLE IV

* McCready-Reeve's t~st for pectic substances.

PectinS

methyl ester

b Na-Pectate

de-esterified

Polygalacturonic acid

de-esterified

+

lA

.Symbols. -:remained clear; +:became brown. 'Saturated distilled water solutions. 1 a . bPolygalacturonic acid methyl ester from Citrus fruits. No. 9135. Polygalacturoni~ acid, sodium salt, water soluble, grade II. No. P18l9.

c ' Polyanhydrogalacturonic acid, grade II, prepared by de-esterification of Citpu8 fruits pectin. No. 9135;.a,b, and c from Stgma Chemical Co. USA.

TABLE V

* ~Cready-Reeve's Pectin test of A. crispa var. mollis nodule sections.

Nodule tissue Host cel! middle Ho'st cel! Endophyte

l'mella cytoplasm capsule .t QI

v'

a Untreated' + Esterified b +

*Symbols, -:remained clear; +:becarne brownb Rand-eut sections of fresh or fixed nodule tissues.

a bAIl pre-fixed sections were hydra~ted before the test. Over-night esterification with 0.5 N HCl in absolute methanol .

..

+

..

. .. ,--

- ,

- 45 -\ /.

capsule was also shown ta stay intact after the treatment. These results

establish the endophyte capsulaI' material as a water insolub