Journal of General Microbiology (rg71), 67,207-214
Printed in Great Britain
Conidium Ontogeny in Penicillium
By J. F L E T C H E R
Botany Department, The University, Newcastle upon Tyne
(Acceptedfor publication I 9 May I 97 I )
SUMMARY
After fixation with permanganate or with glutaraldehyde and osmium tetroxide, phialide walls of Penicillium clavigerum Damelius, P. claviforme Bainier
and P. corymbiferum Westling were electron-transparent with a thin, electronopaque surface layer. The phialide apex was closed by a hollow, plug-like
structure of wall material. A conidium initial was formed by distension of this plug.
The conidium protoplast was delimited from the phialide protoplast by a perforate
septum. A new wall was formed round delimited conidium protoplasts between
the protoplast and the original wall. The original wall round older conidium protoplasts was of different appearance from the original wall around more recently
delimited protoplasts. Conidium chains were covered with a thin, electron-opaque
surface layer. Phialides were uninucleate.
INTRODUCTION
During recent years interest has increased in classifications for Fungi Imperfecti and
particularly Hyphomycetes based on details of spore ontogeny (Hughes, 1953; Madelin,
1966; Barron, 1968). More detailed information on spore ontogeny than has hitherto been
available will probably be required for refining such classification schemes. Conidium ontogeny has been described for the genus Penicillium as a whole from light microscopy (Thorn,
I 9 I 4), for Penicillium corylophilum from time-lapse photomicrography (Cole & Kendrick,
I 969) and for P. clavforme from transmission electron microscopy (Zachariah & Fitz-James,
1967). Zachariah & Fitz-James’s data related, for the most part, to protoplasmic structures.
Further investigation of fine structure is needed, with emphasis on the relationship between
phialide wall and conidium wall. This paper presents results of such an investigation on three
species of Penicillium.
METHODS
Organisms. Penicillium claviforme Bainier (IMI44744), P. corymbiyerum Westling (IMI
62878) and P. clavigerum Demelius (kindly supplied by Dr C. H. Dickinson) were grown on
either malt extract agar or potato dextrose agar in Petri dishes at 2 2 O .
Electron microscopy. Whole coremia of Penicillium claviforme and P. clavigerum, beginning to turn green, were fixed at room temperature by either ( I ) 4% glutaraldehyde in pH 7
Millonig’s phosphate buffer (Pease, 1964) for 3 h., followed by washing with buffer and
treatment with I yo osmium tetroxide in Millonig’s buffer for 3 h., or (2) 1.5yo potassium
permanganate in pH 7 veronal acetate buffer for 1.5 to 16.0 h. Vertical slices, 0.5 mm. thick,
cut from a P. corymbiferum colony where the surface was beginning to turn green were fixed
by method (2) above. Teepol (Shell Chemical Co, London) was added at 0-2yo to glutaraldehyde and potassium permanganate solutions to aid wetting of material and fixation carried
out under vacuum to remove trapped air. Fixed material was washed in buffer, cut into small
pieces, dehydrated, and embedded in Araldite (Ciba-A.R.L. Ltd, Duxford, Cambridge).
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J. FLETCHER
During dehydration, glutaraldehyde and osmium-fixed samples were stained for 30 min.
in 5 youranyl acetate in 70 yo ethanol. Sections (40 to 60 nm. thick) were stained with either
(a) 5% aqueous uranyl acetate for 4 to 5 h. followed by lead citrate (Reynolds, 1963) for
15 min. or (b) Reynolds' lead citrate alone for 30 min. Staining method (a) was usually
used after glutaraldehyde and osmium fixation.
Light microscopy. Whole coremia of Penicillium claviforme and P. clavigerum were fixed
for 10 min. under vacuum in a 6 :I :I mixture of ethanol, glacial acetic acid and lactic acid,
then dehydrated and embedded in paraffin wax. Sections (3 pm.) were cut parallel to the
coremium axis, attached to microscope slides, dewaxed and rehydrated. Preparations of
P. corymbiferum were made by attaching to coverslips strips of double-sided adhesive tape
and pressing the exposed adhesive surfaces on to the surface of a young colony. Penicilli
adhering to the tape were fixed for 10 min. in the ethanol, glacial acetic acid and lactic acid
mixture and rinsed in 90 and 70% ethanol and distilled water. Sections and material on
coverslips were hydrolysed in 1.0N hydrochloric acid for 5 min. at room temperature and for
10to 20 min. at 63".They were then washed in water, followed by phosphate buffer (pH 7),
stained for 30 to 40 min. in Giemsa solution (Ward & Ciurysek, I 962) diluted I :LOin buffer,
allowed to dry and mounted in Euparal (Flatters & Garnett, Manchester).
For phase-contrast microscopy, teased out coremia and small clumps of penicillusbearing hyphae were mounted in aqueous bovine plasma albumen solution (Barer & Joseph,
1955). This reduced the excessive contrast obtained when pure water was used as mounting
medium. The concentration of albumen in which most detail was visible was used.
RESULTS
Electron microscopy
Organelles were sometimes visible in phialide and conidium protoplasts after permanganate fixation (Fig. ~ a c),, but not after glutaraldehyde and osmium fixation (Fig. rd).
Phialide walls, which were similar in all three species, were electron-transparent with a thin,
electron-opaque surface layer (Fig. I a). The distal end of the phialide was closed by a layer
of wall material in the form of a hollow apical plug which, after permanganate fixation, was
often more electron-opaque than was the phialide wall (Fig. Ia). In Penicillium corymbiferum
the plug was thickened below its insertion into the phialide neck (Fig. I C ) . In all three species
conidium initials were formed by distension of the apical plug (Fig. ~b to d ) . The conidium
protoplast was cut off from the phialide protoplast by an initially thin, perforate septum
continuous peripherally with the apical plug (Fig. 2a to c). When fully developed the septum
was thicker but the perforation remained as a channel filled with electron-opaque material
(Fig. 2 4 . Structures which were associated with the septum and which may have been
membranous were observed in P. clavigerum after glutaraldehyde and osmium fixation
(Fig. 2e). In all three species newly delimited conidium protoplasts were enclosed by wall
material derived only from the apical plug and septum (Fig. 2a to e). In P. clavigerum
and P. corymbiferum a new, inner wall layer was present around older conidium protoplasts.
This inner layer was thin around protoplasts close to the phialide and thicker around protoplasts distal from the phialide (Fig. 2f; Fig. 3b). The original delimiting wall around conidium protoplasts distal from the phialide and which developed from the apical plug was thin
and diffuse in appearance (Fig. 3a, b). In P. claviforme the wall material between adjacent
conidium protoplasts distal from the phialide was thicker than the wall between protoplasts
close to the phialide (Fig. 3c). After glutaraldehyde and osmium fixation the wall between
protoplasts distal from the phialide was differentiated into an electron-transparent zone
adjacent to the protoplast and a more electron-opaque zone in the position of the original
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Conidium ontogeny in Penicillium
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Fig. I to 3. Abbreviations: ap, apical plug; CP, conidium protoplast; dw, original delimiting wall;
er, endoplasmic reticulum ;m,mitochondrion; N, nucleus ;nw,new wall layer around conidium protoplast ; p , septa1 pore; pw, phialide wall; s, delimiting septum; sl, electron-opaque surface layer.
Scale markers represent I ,urn. except where stated otherwise.
Fig. I . (a). Phialide and delimited conidium protoplast of Peniciffiumcluvigerum. Permanganate
fixation. (b) Phialide apex of P. cluvigerum with undelimited conidium protoplast. Permanganate fixation. (c) Phialide and undelimited conidium protoplast of P . corymbiferum Pernianganate
fixation. ( d ) Phialide and undelimited conidium protoplast of P . claviforme. Glutaraldehyde and
osmium fixation.
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J. FLETCHER
Fig. 2. (a)Recently delimited conidium protoplast of Penicillium clavigerum. Permanganatefixation.
(b) Recently delimited conidium protoplast of P . cluviforme. Glutaraldehyde and osmium fixation.
(c) Recently delimited conidium protoplast of P. corymbiferum. Permanganate fixation. ( d ) Fully
formed delimiting septum of P. clavigerum. Permanganate fixation. ( e ) Membranous structures
(arrowed) associated with the septum in P. clavigerum. Glutaraldehyde flxation. (f) Phialide
apex and conidium chain of P. clavigerum showingnew wall layer around conidium protoplasts. Permanganatefixation.
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Conidium ontogeny in Penicillium
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delimiting septum (Fig. 3c). In all three species conidium chains were enclosed within an
electron-opaque surface layer (Fig. 3 a to c). After glutaraldehyde and osmium fixation this
surface layer appeared continuous with the surface layer of the phialide wall (Fig. 3 4 .
Light microscopy
Nuclear behaviour appeared to be similar in phialides of all three species. Phialides were
uninucleate (Fig. 3 hl). No fine details of nuclear division could be elucidated. After division
one daughter nucleus was frequently observed in the phialide neck (Fig. 3h,) or in the developing conidium protoplast (Fig. 3h3). The other daughter nucleus remained in the phialide (Fig. 3h,, h3). The spiral arrangement of chromatin prior to division of phialide nuclei
in Penicillium claviforme reported by Zachariah & Fitz-James ( I 967) was not observed here.
By means of phase-contrast microscopy the apical plug in Penicillium corymbiferum
could tentatively be identified as a lighter zone within the apex of the phialide wall (Fig. 38).
In young chains of conidia of P. claviforme and P. clavigerum the region of the delimiting
septum appeared as a darker zone distinct from the conidium wall (Fig. 3e, f).In older
conidium chains the septum region remained only as a narrow connecting strand between
conidia. This was seen most clearly in P. clavigerum (Fig. 3f).
DISCUSSION
Observations reported here reveal formation of a new conidium wall layer after delimitation of conidium protoplasts in Penicillium clavigerum and P. curymbgeerum. The thinner, more diffuse appearance of the delimiting wall around protoplasts distal from the
phialide suggests breakdown of the delimiting wall material as conidia age. In P. clavifurme the greater thickness of wall material between older conidium protoplasts also
suggests that additional wall material is laid down around protoplasts subsequent to delimitation. The greater electron opacity of the septum region between older conidium
protoplasts of P. claviforme could have resulted from breakdown and loss from the septum
of non-staining carbohydrate material which was replaced by the more electron-opaque
embedding medium.
The presence of a connective holding conidia in chains in Penicillium species has long
been known (Thorn, 1914). Phase contrast and electron microscopy of mature conidium
chains has shown the connective in Penicillium griseofulvum to be a thin tubular membrane
within which conidia are linearly arranged and which is distended by each conidium and
constricted between successive conidia (Fletcher, I 969, I 971). This tubular membrane in
P.griseofulvum has an appearance similar to that of the electron-opaque surface layer around
conidium chains of P. clavvorme, P. clavigerum and P. corymbiferum reported here. No
mature conidium chains of P. clavigerurn, P.claviforme and P. corymbiferum were seen in
the electron microscope, probably because they were too fragile to withstand preparation
procedures. However, electron micrographs of younger chains showed that total breakdown
of the wall derived from the apical plug, leaving conidia enclosed in the electron-opaque
surface layer, would have given an arrangement similar to that found by Fletcher (1969,
1971) in P. griseofulvum. The connective would then have consisted solely of the electronopaque surface layer.
The mode of conidium formation in Penicilliuin clavigerum, P.claviforme and P. corymbiferum which is proposed from the findings of the present electron microscope study is summarized diagrammatically in Fig. 4. The evidence from phase-contrast microscopy that the
region of the delimiting septum is distinct from the conidium wall and remains only as a
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Conidium ontogeny in Penicillium
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narrow connecting strand between mature conidia supports this proposed mode of conidium
formation. The origin of the apical plug has not been ascertained in this investigation. Cole
& Kendrick ( I 969) suggested that a layer of wall material, which would become the apical
plug according to the mode of conidium formation proposed here, is laid down within the
still intact phialide apex before formation of the first conidium protoplast. Hyphomycete
genus Aspergillus formation of a new wall layer and breakdown of the delimiting wall
have not been observed, but fine structure of conidium delimitation is probably similar to
that in Penicillium (Tanaka & Yanagita, 1963; Trinci, Peat & Banbury, 1968).
The genus Penicillium is usually placed in group IV of Hughes’s (1953) classification
scheme (Madelin, 1966; Barron, 1968). The mode of conidium ontogeny proposed here
fits the revised definition of group IV given by Cole & Kendrick (1969). However, formation
sl
.# I-
P
A
Fig. 4.Proposed mode of conidium formation in Penicillium. A to D. Formation and delimitation
of a conidium protoplast at the phialide apex. E. Conidium chain showing stages in formation of
mature conidia by development of a new wall layer around protoplasts and breakdown of the delimiting wall. Abbreviations: ap, apical plug; c, connective; CP, conidium protoplast: dw, delimiting
wall; nw, new wall around conidium protoplast; p , septa1 pore; p w , phialide wall: s, delimiting
septum; sl, electron-opaque surface layer.
Fig. 3. (a) Distal part of a conidium chain of Penicillium clavigerum showing diffuse appearance
of original delimiting wall and the electron-opaque surface layer. Permanganate fixation. (6) Part
of a conidium chain of P. corymbiferum showing new wall layer aroundconidium protoplasts, diffuse
appearance of the original delimiting wall and the electron-opaque surface layer. Permanganate
fixation. (c) Phialide apex and conidium chain of P. claviforme showing the greater thickness
and differentiation into two zones of wall material between conidium protoplasts distal from the
phialide, and the electron-opaque surface layer. Glutaraldehyde and osmium fixation. ( d ) Phialide
apex of P. clavigerum showing continuity (arrowed) between electron-opaque surface layers of
phialide and conidium chain, Glutaraldehyde and osmium fixation. (e) Phialides and young
conidium chains of P. clavigerum. The region of the delimiting septum (arrowed) is distinct from
the conidium wall. Phase contrast microscopy. Scale marker = 5 pm. (f)Phialides and young
conidium chains, and a longer detached chain of older conidia, of P. claviforme. The region of
the delimiting septum (arrowed) is distinct from the conidium wall. Phase contrast microscopy. Scale
marker = 5pm. (g) Phialides and young conidium chains of P. corymbiferum showing a lighter
zone within the phialide apex in the position of the apical plug. Phase contrast microscopy. Scale
marker = 5 pm. (h) Phialide nuclei of P. cluviforrne. Scale marker = 5 pm.
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J. FLETCHER
of a new wall layer within a delimiting wall, breakdown of the delimiting wall and retention
of conidia within a membranous connective are features not used in Cole & Kendrick‘s
definition. It remains to be seen whether or not these features are characteristic of other
fungi referred to group IV of Hughes’s scheme. From light microscope evidence these
features do not appear to be present in Thielaviopsis basicola (Brierly, 1915; see Cole &
Kendrick, I 969, for nomenclature), Phialophora Zagerbergii and the Thielaviopsis state of
Ceratocystis paradoxa (Cole & Kendrick, 1969) which are referred to group IV (Hughes,
1953; Barron, 1968) and have phialides which are larger and more easily observed by light
microscopy than those of Penicillium.
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