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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. ~
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\) 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.
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Suggested short title
o
Alnus root nodul~ endophyte.
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Maurice Lalonde .,
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---,-__ --- - --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 .
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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
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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
, ,
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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 "
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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
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PAGt 82
82
86
87
94
99
~ 102
103 L
105
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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.
" .'
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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 '
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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.
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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
\,
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xix
PAGE 0
92
92
92 '
93
93
93
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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.
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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
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