399
Mycol. Res. 94 (3):399-406 (1990) Printed in Great Britain
Cytology of the life-cycle of Morchella
THOMAS J. VOLK* A N D THOMAS J. LEONARDt
*Department of Botany and t Departments of Botany and Genetics, University of Wisconsin-Madison, Madison, WI 53706, U S A .
Cytology of the life-cycle of Morchella. Mycological Research 94 ( 3 ) :399-406 (1990).
Various stages of the morel life-cycle are studied cytologically. Photomicrographic evidence demonstrates that the average number
of nuclei per cellular compartment in vegetative hyphae of Morchella is 10--15 and that hyphal fusions are quite frequent. The
resting structures, the sclerotia, are actually pseudosclerotia which form from the repeated branching and enlargement of terminal
hyphae horn either primary (homokaryotic) or secondary (heterokaryotic) hyphae. Photomicrographs also depict the development of
fruiting body primordia. Photomicrographs of ascus development demonstrate autogamy rather than de novo heterokaryon formation
by hyphal fusion in the subhymenial layer of the fruiting body. For the first time a comprehensive life-cycle diagram of the morel is
introduced.
Key words: Morel, Morchella, Cytology, Life-cycle.
Despite obvious commercial applications and general interest
in the morel (Morchella spp.), relatively little has been published
on this fungus. One area of neglect is the cytology of various
developmental stages of the Morchella life cycle :the vegetative
hyphae, sclerotia, primordia, and fruiting bodies. Presumably
this is due to the general scarcity of the fruiting bodies both
in nature and in the laboratory.
The most extensive cytological study of Morchella was
performed by Greis (1940) in an interesting but controversial
series of studies with M. conica, M. esculenta, and M . elata,
three species of Morchella collected from the wild, in which he
traced-the path of nuclei from vegetative hyphae through
various tissues of the fruiting body, progressing from the
sterile cells, through the subhymenial layer, and into the
hymenium. He reported plasmogamy occurred by somatogamy in M . conica and M . esculenta among the cells of the
subhymenial or the hymenial layers and by autogamy in
M. elata, where two nuclei from a single multinucleate cell were
delivered to the ascus mother cell of the hymenium. Greis did
not state whether plasmogamy occurred in M. elata or
whether the nuclear condition of the cells precluded the
necessity of plasmogamy, although no sex organs were
observed. Our own observations are at some variance with
Greis's cytological interpretations in that numerous vegetative
strains of the morel in our studies exhibited extensive
anastomosis and there is even some evidence for heterokaryon
formation (Volk & Leonard, 1989).
Ower (1982) studied fruiting-body development in M.
esculenta from very small primordia and reported the fruiting
t To whom correspondence should be addressed.
body develops from a single cell. No evidence was provided
to support this view, and it contrasts with Gessner, Romano
& Schultz (1987) who claim the results of their isozyme study
are more consistent with fruiting-body development from a
conglomerate of sterile haploid tissue.
Despite controversy on fruiting initiation, other aspects of
fruiting morphogenesis, the developmental cytology of the
ascus, remain clear; the nuclear divisions in the ascus mother
cell and the ascus have been previously described (Maire,
1904, 1905).
Although the ascospores shed by the fruiting bodies are
easily collected in large numbers and germinate readily
(Schmidt, 1983), the vegetative mycelium arising from the
spores has not been characterized. The vegetative mycelium
may be quite extensive and may form sclerotia. These
structures were first reported in the morel by Molliard (1905)
who observed that their formation could occuf on 'sterile
moistened bread' and that they could grow to be quite large.
He did not, however, realize the significance of sclerotia in the
morel life-cycle. This appreciation did not occur until Ower
(1982) used sclerotia as a nutrient sink under 'defined'
conditions to observe the first occurrence of controlled
fruiting in Morchella esculenta.
The present study examines the progression of cytological
events in the morel life-cycle, including morel fruiting-body
development, the development of ascospores in an ascus,
vegetative mycelia, heterokaryon formation, sclerotium formation, sclerotium germination, and primordium development,
and introduces for the first time a composite life-cycle
diagram, based on the findings of this study and published
literature, outlining a sequence of developmental events from
spore to spore.
Experimental studies on the morel
MATERIALS A N D M E T H O D S
In the absence of cultivated ascocarps, we collected 'wild'
fruiting bodies of Morchella esculenta Pers., which were
immediately stored in plastic bags at 4 OC. Ascospores were
collected by bisecting the fruiting bodies and allowing them
to eject their spores en rnasse onto sterile Petri dishes.
Vegetative cultures of Morchella that were derived from these
spores were grown on CYM medium, a complete medium
plus yeast extract (Leonard & Dick, 1973). This medium plus
2 % composted sheep manure (obtained locally) was found to
be favourable for sclerotium formation. Some fruiting bodies
of Morchella were obtained from chance fruitings in association
with tuberous begonias (Begonia tuberhybrida) under semicontrolled conditions, although most were obtained from the
wild.
Some specimens were stained with dyes that cause nuclei to
fluoresce. The first of these were living specimens mounted
directly in 0.0025% acridine orange in veronal acetate
(Yamomoto & Uchida, 1982). The second involved hydrolysis
in 4 N-HCl followed by mounting in 150 pg/ml acriflavin in
K,S,O, (5 mg/ml in 0.1 N-HCl) (Raju, 1986); the third
method consisted of fixation in 5 % glutaraldehyde in 0.067 m
phosphate buffer (pH 7.0) followed by staining in 500 ug/
ml mithramycin in 20 mg/ml mannitol in the same buffer
(Heath, 1980). Cytology of these specimens was carried out
with a Zeiss epifluorescence microscope (excitation filter
465-495, barrier filter LP 515, dichromic reflector FT 510).
Other specimens were fixed in 3 : 1 ethano1:acetic acid,
hydrolysed in hydrochloric acid and stained with iron
haematoxylin in propionic acid (Henderson & Lu, 1968) or
Giemsa stain (Elmallah & Pijnacker, 1979) and observed using
optical microscopy.
RESULTS
Fruiting-body morphogenesis
The most familiar and conspicuous stage of the morel lifecycle is the fruiting body, which represents a very advanced
ascomycete reproductive structure. It consists of many
apothecia arranged all over the surface of the conical spongelike structure which are collectively raised above the ground
so that spores can be released and effectively caught in air
currents for dispersal. We have repeatedly, although not under
controlled conditions, observed the development of morel
fruiting bodies in incidental culture with begonias (Begonia
fuberhybrida), from tiny primordia to mature fruiting bodies
(Fig. 1). The primordium is at first a tiny white finger-like mass
of hyphae. The hymenium differentiates at the very apex,
enlarging and pigmenting as the primordium grows into a
mature fruiting body. The most mature fruiting body in this
series is medium size, but we have obtained larger structures
in our begonia beds.
Ascus development
Because begonia-associated morel fruiting was not predictable
or controllable in any way, we also made cytological
observations with fruiting bodies of M . esculenta collected
Fig. 1. Development of Morchella fruiting bodies in culture with
begonia plants (Begonia fuberhybrida). Direction of development of
the fruiting body is from right to left in this picture. Photo is actual
size.
from the wild which were allowed to mature in the refrigerator
at 4'. Although typical expansion of the fruiting bodies did
not occur after collection, the nuclei of many asci were found
to undergo mitotic divisions, karyogamy, and meiosis while
refrigerated. Subsequent post-meiotic mitoses and ascospore
delimitation were also observed. Thus, refrigeration provides
convenient storage and allows continued progression of
developmental events associated with the nuclear cycle in asci.
This convenience eliminates having to guess the order of the
morphogenetic and cytological stages, as would be necessary
with a series of fruiting bodies as carried out by Greis (1940)
in a previous study.
Observations of the refrigerated fruiting bodies revealed
that the ascus mother cell is multinucleate with a large number
of nuclei (Fig. 2A). Two of the nuclei, already paired, migrate
to the tip of the ascus mother cell, and a large vacuole forms
behind them (Fig. 2B). These two nuclei fuse to form a large
diploid nucleus (Figs. 2C, 3) with the large vacuole remaining
in place. No hook cells or croziers were observed. Meiosis
occurs, followed by four successive mitotic divisions, and the
ascospores are delimited by free cell formation as described
for Ascobolw by Oso (1969). After the ascospore walls form,
the mature ascus contains eight ascospores (Fig. 4), and we
observed each of the eight mature ascospores contains eight
nuclei as reported by Maire (1905). The ascus may be
described as a typical unitunicate operculate-type ascus, with
eight ascospores that are explosively released and may travel
up to several metres (Schmidt, 1979). The spores exit through
an opening created by the rupture of a hinged lid-like
structure, the operculum (Samuelson, 1978). Each ascospore
germinates within a few days under natural conditions
(Schmidt, 1983) or on simple media to form a mycelium, which
we found consists of a single nuclear type (Volk & Leonard,
1989).
T. J. Volk and T. J. Leonard
Fig. 2. Ascus development in Morchella. Acridine orange stain. A,
Multinucleate proascus is very dense with nuclei. B, Two of the
nuclei which were already observed to be paired migrate to the tip
of the ascus mother cell, followed by the formation of a large vacuole
behind them. C, These two nuclei undergo fusion to form a very
large fusion nucleus which is considerably larger than either haploid
nucleus. Bar = 5 urn. Fig. 3. Fusion nucleus in ascus mother cell. Iron
haematoxylin stains the nucleolus under these conditions rather than
the nucleus, which appears translucent and irregularly shaped.
Bar = 5 urn.
Vegetative hyphae
The monosporous vegetative hyphae of Morchella are rapid
colonizers when grown on CYM or other nutrient agar plates,
hlly colonizing an 8-5 cm Petri dish within 5 - 6 d (average
growth rate 0.4-0.5 mm/h) at room temperature (22-25').
Growth is still relatively prolific at lower temperatures; the
same large Petri dish may be colonized in 12-15 d at 4'.
Generally when morel mycelium colonizes a substrate, a dark
brown pigment is secreted into the medium, first From the
older mycelium in the centre of the colony and progresses to
the periphery of the plate with ageing, giving the Morchella
colony a characteristic brownish appearance. When viewed
microscopically, the relatively large diameter of the hyphae
(5-10 pm), their branching patterns, and their tendency to
anastomose frequently (Fig. 5), provide along with the
Fig. 4. Mature asci of Morchella containing eight ascospores each.
Ascospores are generally linearly arranged, but are easily jumbled in
the ascus during isolation. Bar = 5 um.
macroscopic characteristics, a set of traits that collectively
typify morel mycelia.
The hyphal compartments of all the Morchella species
examined are multinucleate and more so than reported by
Greis (1940). Nuclei were visualized in the present study using
various nuclear stains that included propionic iron haematoxylin (Fig. 6), as well as Giemsa, acridine orange, mithramycin, and acriflavin. Different stains were used to corroborate
the fact that each of them was indeed staining nuclei. The
multinucleate condition was also studied with phase microscopy (Fig. 7) using hyphae grown from spores on a thin
layer of agar under a coverslip. This method restricted the
formation of vacuoles which under normal conditions obscure
the nuclei. Staining with acridine orange in veronal acetate
was particularly useful for visualization of nuclei because of
the dye's bright fluorescence and also because it was so easy
to use. Since RNA also fluoresces with this stain, although
much less so, its diffusiveness allowed the outer limits of the
'cells' to be observed, and this provided some contrast which
facilitated the counting of nuclei in hyphal compartments.
Most compartments average 10-15 nuclei (data not shown),
with a wide range of variation: the fewest number were found
in newly-partitioned tip 'cells' (1-2 nuclei), while sub-terminal
'cells' showed the average 10-15 per cell (data not shown;
see also Fig. 6). The septa1 delimitations of the compartments
consist of simple porate septa which may allow passage of
some organelles, including nuclei, and could account for the
variability of nuclei per cell. Compartments with 40-50 nuclei
are not uncommon; an extreme example of this multinucleate
state was a single compartment with 65 nuclei.
Heterokaryon formation
When compatible primary mycelia of Morchella anastomose
they are capable of forming a limited albeit stable hetero-
Experimental studies o n the morel
Fig. 5. Morchella hyphae have a distinct tendency to anastomose.
This monoascosporic mycelium was grown on a cellophane
membrane, floated, and mounted on a slide in water. It is
representative of mycelia on other media. Bar = 5 pm. Fig. 6. The
hvphae of Morchella are multinucleate with the cellular com~artments
each containing an average of 10-15 nuclei. Propionic iron
haematoxylin stains the nuclei a dark colour. Bar = 5 pm. Fig. 7.
Multinucleate hyphae unstained. Phase contrast. The nucleus appears
as a translucent area with a dark nucleolus in the centre (arrow).
Bar = 5 pm.
402
Fig. 8. Microscopic examination of heterokaryon from genetic
complementation test. Nuclear pairs such as these can be seen in most
of the hyphae. Subcultured hyphae from fruiting-body stalk cells and
from mycelial melds show similar nuclear pairing. Bar = 5 pm.
- a
karyon. Some genetic and cytological aspects of this
phenomenon have been reported (Volk & Leonard, 1989).
Such hyphae can exhibit genetic complementation, and
staining with mithramycin reveals some nuclear pairing
(several pairs per cell) resembling dikaryons (Fig. 8). Such
distinct nuclear pairing does n o t occur frequently in
monoascosporous mycelium, and never occurs more than
once per single cell of the monosporous culture. This nuclear
pairing can also be seen in certain sclerotia and in the sterile
cells of the fruiting body, providing a possible link between
the vegetative heterokaryon, the heterokaryotic sclerotium,
and fruiting-body formation.
Sclerotium formation
When the mycelia of Morchella are grown at low temperature
(4') o n CYM plus 2 % composted sheep manure, or when
nutrient depletion occurs (such as complete colonization of a
Petri dish), they usually form sclerotia. These are not true
sclerotia in the classic sense of Sclerofinia sclerotiorum, which has
sclerotia that differentiate the complex tissues of the rind and
the medulla, but rather they are the undifferentiated
'pseudosclerotia' characteristic of Monilinia frucfigena. Willetts
(1972) refers t o any 'macroscopic fungal resting structure' as
a sclerotium, and w e will adopt this terminology for the morel.
The sclerotia of Morchella are easily and rather rapidly
formed on CYM containing 2 % sheep manure. After 7-10 d
growth o n this medium, small (1-2 mm) sclerotial initials
begin t o form and expand, usually coalescing to form a single
large sclerotium. The sclerotia are considered mature when
T. J. Volk and T. J. Leonard
Fig. 9. Initiation of sclerotia on CYM plus 2% composted sheep manure medium. Sclerotia are initiated from the repeated profuse,
compacted branching of a terminal hypha. Bar = 5 vm. Fig. 10. Young sclerotium initial. Note rounded cell shape which differs
significantly from that of the vegetative hyphae. Unstained. Bar = 5 urn.
radial expansion ceases and the pigmentation attains a dark
brown colour. This laboratory method also provided the
material for studying the cytology of sclerotium development.
The sclerotia of Morchella are of the terminal type, as
defined by Willetts (1972),formed from the repeated branching
of a terminal hypha (Fig. 9). The cells 'round up' to form
varied and unusual shapes with thick walls (Fig. 10) and
remain multinucleate. Physiologically, the sclerotia begin to
store nutrients, some as lipids, as suggested by the
accumulation of oil droplets which are easily observed.
Finally, when the sclerotia are mature, a cross-section reveals
a series of compacted, isodiametric cells with very thick walls.
At this stage the sclerotium can tolerate adverse conditions
such as low temperature and desiccation.
Carpogenic and myceliogenic sclerotium germination
When the sclerotia of Morchella germinate, they appear to
have two options and resemble Sclerofinia spp. (Willetts, 1972)
in this regard. They may form a new vegetative mycelium
(myceliogenic germination) which is morphologically and
cytologically similar to the pre-sclerotium vegetative mycelium, or they may form a fruiting body (carpogenic
germination). In carpogenic germination the fruiting body
does not emerge directly from the sclerotium as it does in
Sclerotinia or Monilinia (Willetts, 1972), but develops from
hyphae that emanate from the sclerotium.
Ower (1982) provides photographs of macroscopic fruitingbody morphogenesis in culture which closely resemble our
begonia bed morels. We have also observed primordium
formation in agar culture; the first indication of fruiting-body
formation is a tuft of light-coloured mycelium above the
substratum (Stage I ~rimordium;Fig. 11). The fruiting-body
primordium emerges from the centre of this tuft which
consists of a series of tightly cohering and roughly parallel
hyphae (Stage I1 primordium; Fig. 12). This stage is particularly
vulnerable to abortion, but under optimal conditions fruitingbody formation may continue as in Fig. 1.
DISCUSSION
When all phases of the morel life cycle from micro- to
macroscopic observation are considered, it is found to be
largely similar to other higher ascomycetes. The limited
nuclear pairing in vegetative heterokaryons, established by
plating complementing drug resistant monoascosporous
mycelia (Volk & Leonard, 1989), is an interesting contrast to
other ascomycetes if this pairing represents a significant phase
in heterokaryons of the morel. We have no firm evidence that
nuclear pairing is a natural phase of the morel life cycle or
even that the two pairing nuclei are in fact different. Such
pairing is, however, a common feature of forced heterokaryotic
mycelia in Morchella and is also found in mycelia from
subcultured fruiting-body stalk cells, and in the interaction line
of confluence between monoascosporous isolates (mycelial
melds) (Volk & Leonard, 1989). All of these mycelia are
indirectly or directly associated with carpophore formation.
The relationship of all these developmental phases may be
summarized in a life cycle diagram (Fig. 13) that is consistent
with our observations as well as the published literature.
Certain assumptions have been made with regard to some
developmental details and position of specific growth phases,
to bridge laboratory observations with what is thought to
happen in nature.
The typical morel fruiting body develops over the course
of several days in the spring and forcibly ejects its ascospores
(Schmidt, 1979). We h d the ascospores germinate almost
immediately on any nutrient medium (see also Schmidt, 1983).
The hyphae are multinucleate, averaging 10-15 nuclei per
compartment, although we have observed compartments with
Experimental studies on the morel
Fig. 1%. Fruiting-body primordiurn Stage I. Light-coloured tufts of
mycelium emerging from a sclerotium on agar medium. Bar = 5 vm.
Fig. 12. Fruiting-body prirnordium Stage 11. Aggregation and
cohesion of aerial hyphae into a pin-shaped fruiting-body initial. No
further development was observed in these cultures. Bar = 5 pm.
as many as 65 nuclei. It is difficult to rationalize the value of
such multinucleate cells. In other ascomycetes with porate
septa such as Neurospora, the nuclear number is also variable
in a cellular compartment from I to 15 depending on the
conditions, while in Venturia the nuclear number is quite stable
at one or two per cellular compartment (Fincham, Day &
404
Radford, 1979). The morel may prove to be exceptional
among the ascomycetes with regard to multinucleate
condition,
Another distinctive feature of Morchella hyphae is frequent
anastomosis with one another. The frequent occurrence of
hyphal fusions, even in a monoascosporous colony, contrasts
with many other fungi. Although the reason for such a high
frequency of fusions among morel hyphae is not known, it
may be related to sexual reproduction, which depends on the
formation of large fruiting bodies and may involve substantial
channelling of nutrient reserves into the developing ascocarp
(Thrower & Thrower, 1968a, b). After fusion the cytoplasms
of the two cellular compartments become continuous allowing
free and readily observable passage of nuclei and other
organelles.
Interspecific hyphal fusion occurs between some species of
Morchella (M. esculenta Pers., M . crassipes (Vent.) Pers., and M .
deliciosa Fr.) but does not occur between other species (M.
semilibera QuC1. and M . angusticeps Pk). In fact these latter two
do not undergo hyphal fusion with any of the other morel
species in this study. These observations lend credence to the
concept (Nancy Smith Weber, pers. comm.) that M. esculenta,
M . crassipes, and M . deliciosa are ecotypes of the same species,
and that M . semilibera and M. angusticeps are in fact different
species.
There are two possible pathways in the morel life-cycle
subsequent to vegetative growth; these two pathways differ
primarily at the point at which plasmogamy takes place. Path
1 leads directly, without plasmogamy, from the primary
mycelium to sclerotium formation, when conditions do not
favour further vegetative growth, e.g. poor nutrition, lack of
moisture, adverse temperature, etc. Such induction by adverse
conditions is not the exclusive stimulus, as sclerotia can
develop in response to certain nutrients as well, such as
composted sheep manure. Nevertheless, whatever the driving
force, once the commitment is made to initiate sclerotium
development, the growing portion of the primary mycelium is
induced to round up and to form the thick, darkly-pigmented
walls that are characteristic of a sclerotium. These primary
sclerotia may overwinter and may germinate carpogenically in
the spring to form the fruiting hyphae. According to Ower
et al. (1986), a single mycelium of this type can produce the
typical fruiting body, although our data (Volk & Leonard,
1989) suggest that fruiting is not limited to primary mycelium.
If not properly conditioned, i.e. not given the proper
environmental or nutritional signals, the primary sclerotia can
germinate myceliogenically to form new primary mycelia and
again grow vegetatively as is the case with Sclerotinia and
other species.
If there is interaction with another compatible primary
mycelium, Path 2 may ensue. We have observed that when
hyphae of two genetically different mycelia interact, heterokaryotic hyphae may arise. This second mycelium is
represented in culture by the formation of an aerial ridge of
hyphae with the deposition of dark pigment at the line of
confluence. We refer to this as the 'mycelial meld', a term
coined specifically to describe this reaction because of the
melding or fusion of the aerial mycelia (Volk & Leonard,
1989). If conditions become unfavourable for further growth,
T. J. Volk a n d T. J. Leonard
405
Fig. 13. Proposed flow diagram of developmental events in the life-cycle of Morchella. Beginning with the asci, which are unitunicate
and ordered. the ascospores are forcibly ejected and readily germinate to form a primary mycelium with numerous scattered nuclei.
There are two alternate pathways, path 1 and path 2, which differ depending on whether heterokaryosis occurs. If conditions are
appropriate, path I may ensue; the primary mycelium can form sclerotia to survive adverse conditions such as winter. In the spring the
sclerotium may germinate carpogenically to form a fruiting body (according to Ower ef al., 1986), or it may germinate myceliogenically
to form a new primary mycelium. Alternatively path 2 is followed if a primary mycelium meets another compatible primary mycelium;
the two hyphae fuse to form a heterokaryon with paired nuclei. This heterokaryotic mycelium may also form sclerotia for
overwintering. In the spring presumably these sclerotia also have two options for germination: myceliogenic or carpogenic. The results
of these studies and of published data d o not rule out the existence of either pathway; it is possible that either or both may occur
under natural conditions. Drawings not to scale.
Ascus with ascospores
Spore release
I
f
.
Germ~nat~on
Carpogenic
germination
''\ \
\
Myceliogenic
germination
Experimental studies o n the morel
the heterokaryotic hyphae may form a heterokaryotic
sclerotium morphologically similar t o the one used for
overwintering in nature. After any 'conditioning' effects of
freezing and thawing associated with the winter and early
spring, the heterokaryotic sclerotium of Path 2 also has t w o
options for germination: myceliogenic germination, which
regenerates a secondary mycelium, and carpogenic germination, which leads t o fruiting-body formation. These
alternatives between vegetative growth and reproductive
development are also characteristic of other sclerotiumforming fungi as Sclerofinia and Monilinia (Willetts, 1972).
This study provides the first attempt a t a cohesive, working
version of the life-cycle of M o r c h e l h based o n the results of
this study and published literature. It is not intended to be the
final work o n this subject, but it is hoped that it will stimulate
further discussion o n the biology of Morchella and provide
much of the framework that will be necessary for the
manipulation of the morel life-cycle.
The authors wish t o thank Dr John F. Leslie for critical
reading of the manuscript and helpful suggestions. We also
thank Kandis Elliot for illustration of the life-cycle.
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