Mycol. Res.

102 (6) : 719735 (1998) Printed in Great Britain 719

The role of morphogenetic cell death in the histogenesis of the

mycelial cord of Agaricus bisporus and in the development of
macrofungi

M. H A L I T U M A R A N D L E O J. L. D. V A N G R I E N S V E N
Mushroom Experimental Station, P.O. Box 6042, 5960 AA Horst, The Netherlands

Hyphal growth of the white button mushroom Agaricus bisporus on spawn grains and compost is typically vegetative. Hyphae are
loosely arranged, in contrast to the organized texture in tissues, and needle-like calcium oxalate crystals are frequently present on the
surface of vegetative hyphae. The mycelial cord is the first well-organized tissue of the fruiting mycelium ; it is surrounded by fluffy
white hyphae that grow vegetatively. The hyphae of the cord are held together through a semi-fluid medium, the extracellular
matrix, which aids in creating a three-dimensional pseudoparenchymatous structure. The matrix material seems to be secreted into
the extracellular environment by specifically differentiated cells, but the vegetatively growing hyphae of A. bisporus initially exploit a
different mechanism in the production of matrix which involves a type of cell death different from cell necrosis. This primary matrix
production leads to the formation of minute cord tissues in which oxalate crystals are no longer present. Once the hyphal cells of A.
bisporus pass the threshold from a vegetative form into organized structures, they become differentiated and self-maintaining in the
production of the extracellular matrix material. Morphogenetic cell death has been observed before in A. bisporus development and
here we show that it occurs in various species of macrofungi : a mucoid zone of the pileipellis typically found in developing fruit
bodies of Psilocybe and Panaeolus spp. contains numerous, dying or dead hyphal cells which show ultrastructural features comparable
to those observed during the mycelial cord formation of A. bisporus. Studies performed using specimens of Stropharia rugoso-annulata,
Coprinus domesticus, Psathyrella candolleana, Tremella mesenterica, Otidea onotica and Peziza ostracoderma in representative growth stages
revealed supporting evidence for the view that morphogenetic cell death plays a key role at different stages during the development
of fungal fruit bodies. This phenomenon may be related to the programmed cell death occurring in developing plants and animals.

The cultivated edible mushroom of Agaricus bisporus has a
great commercial value. Understanding morphological aspects
of its growth greatly assists maintenance of a healthy crop.
From a morphological point of view, the mycelial cord is
considered to be the first fungal tissue and has different
properties from the solely vegetatively growing hyphae in the
compost. Under specific conditions of cultivation (Van Gils,
1988), the formation of mycelial cord is an essential prerequisite
to fruiting. If the mycelial cords are not formed the primordia
do not appear and the mycelial growth continues with an
excess amount of fluffy white hyphae (FWH) that eventually
cover the surface of the culture bed.
Since macrofungi are built of hyphae, these branching
elements are the basic architectural blocks of the cord tissue
and the developing fruit bodies (Carlile, 1995). Vegetatively
growing, widely separated and overlapping hyphae are
apparently not capable of fulfilling the function of basic
building blocks of fungal tissues and organs. We have recently
demonstrated that fungal tissues are formed by an orderly
arrangement of hyphae within a ground substance known as
extracellular matrix (ECM) (Umar & Van Griensven, 1997 a).
A transformation from vegetative growth into tissue formation
is a crucial change in the fungal development during which the
ECM must first be produced. The present investigation is
primarily aimed at determining the morphological events
leading to the histogenesis of the mycelial cord during the
cultivation of A. bisporus.
Early stages in cord formation seem to involve a type of
programmed cell death and our earlier studies on fruit body
development also revealed that morphogenetic cell death
plays a major role in the development of the gill tissue and
hymenial chamber in A. bisporus (Umar & Van Griensven,
1997 b). The general importance of cell death in fungal
development is illustrated here by observations on Psilocybe
cubensis, Panaeolus cyanescens, Stropharia rugoso-annulata,
Coprinus domesticus, Psathyrella candolleana, Volvariella bom-
bycina, Otidea onotica and Peziza ostracoderma. In all developing
fruit bodies studied here, we observed morphogenetic cell
death more than once in various tissues and organs.
MATERIALS AND METHODS
Rye spawn grains, colonized with A. bisporus (J. E. Lange)
Imbach, strain Horst2 U1 were obtained from Sylvan Spawn
(Horst, The Netherlands). Colonized compost after phase 2
consisting of a fermented mixture of wheat straw, chicken and
horse manure and gypsum, and casing soil consisting of a
10 : 1 mixture of black peat and lime chalk (calcium carbonate)
Morphogenesis and programmed cell death in fungi 720

were obtained from CNC (Milsbeek, The Netherlands).

Mycelial cords, gathered from the casing soil in our cultivation
units, and spawn grains were processed for LM, TEM and
LTSEM after gross anatomical examination as described by
Umar & Van Griensven (1997 a). An aqueous solution of both
toluidine blue O (1 % w}v) and crystal violet (01 % w}v) was
used to stain the 2 m thick tissue sections. For fluorescence
microscopy, an aqueous solution of acridine orange (001 %,
w}v) was applied to the sections for 15 s. The stained sections
were thoroughly washed with distilled water, and dried on a
hot plate to avoid the decolorizing effect of alcoholic
solutions. Some ultrathin sections were treated with the usual
PAS staining as described for histopathological practice (Lillie
& Fullmer, 1976) with and without previous digestion using a
1 % diastase solution (mixed and amylase ; BDH Chemicals, Fig. 2. Penetrating hyphae between the layers of the husk. Note the
Prod. No. 39013, Poole, England). crystals lying almost perpendicularly to the long axis of the hyphae.
Stropharia rugoso-annulata Farl. ex Murrill was cultivated Toluidine blue staining. Scale bar, 20 m.
using wetted straw as substrate and Agaricus casing soil.
Psilocybe cubensis (Earle) Singer, Panaeolus cyanescens Berk. &
Broome, Volvariella bombycina (Schaeff. ex Fr.) Singer were
cultivated as described for A. bisporus (van Gils, 1988).
Ascomycetous fungi Peziza ostracoderma Korf and Otidea
onotica (Pers.) Fuckel, which grew in the trays during the
cultivation of Stropharia, and Coprinus domesticus (Bolt, ex Fr.)
Gray, Coprinus sp., Psathyrella candolleana (Fr.) Maire, Tremella
mesenterica Retz. ex Hook, which were collected in the fields,
were included in this investigation.

RESULTS
Spawn grains were found to be almost entirely covered by a
whitish layer of hyphae of A. bisporus. Histological sections of
the grains showed diffuse mycelial growth on the surface, and Fig. 3. Detailed view from a similar location showing needle-like
crystals. Slightly polarized light. Toluidine blue staining. Scale bar,
in and between the layers of the husk, penetrating towards the
10 m.
centre alongside or through the aleurone cells (Fig. 1). The
hyphal arrangement was loose and the hyphae contained
many needle-like crystals on the cell walls (Fig. 3). About one
day after inoculation with spawn, mycelial growth started in

Fig. 4. Hyphae of A. bisporus growing vegetatively in the inoculated

compost. Note the three-dimensional spread of the hyphae bridging
enormous distances between the straw particles. Scale bar, 10 mm.

Fig. 1. Cut surface of a rye grain grown with the mycelium of A.

bisporus Horst2 U1. LM. Toluidine blue staining. Mycelium has the compost on the pieces of straw as a white, fine, and usually
covered the surface and has penetrated into the grain through the radially expanding network of hyphae. The samples taken
layers of the husk. Note the granular and metachromatic staining of after a complete colonisation of the compost demonstrated
the aleurone cells. Scale bar, 100 m. that hyphae had invaded the straw cells by growing through
M. Halit Umar and Leo J. L. D. Van Griensven
Fig. 5. Following the application of the casing soil on the full-grown
compost, ascending mycelial cords are formed. They are frequently
interconnected. Scale bar, 10 mm.
Fig. 6. A mycelial cord of Stropharia rugosoannulata. Note the
primordium and mycelial cord covered by FWH. Scale bar, 3 mm.
Fig. 7. LM of a newly formed mycelial cord of microscopical size.
Note the small globules between the hyphae of the universal veil
enveloping this minute cord tissue. Crystal violet staining. Scale bar,
300 m.
their walls (Fig. 22). Large air spaces between the pieces of
straw were crossed by hyphae with apparent ease (Fig. 4).
Hyphae originating from the grains grew in all directions
including downwards in the 20 cm thick bulk of compost,
721
Fig. 8. Detailed view of the sky-blue globules between the hyphae.
They seem to be amorphous structures in LM. Crystal violet staining.
Scale bar, 50 m.
Fig. 9. Higher magnification of the globules between the hyphae.
These blue bodies physically connect the hyphae together. Crystal
violet staining. Scale bar, 20 m.
Fig. 10. A 2 m thick section of the mycelial cord of A. bisporus
stained with acridine orange. Fluorescence microscopic image.
Contrary to the bright yellow fluorescence of the chitinous wall of
the hyphae, the globules are orange-green, sharply delineated and
always show a connection with some hyphae at one point. Scale bar,
25 m.
indicating insensitivity to gravity. Under the right climatic
conditions the compost became diffusely colonized by the
mycelium within 2 wk of spawning. If compost fully penetrated
by mycelium was not covered by casing soil, no primordia
Morphogenesis and programmed cell death in fungi 722

Fig. 11. Bluish globules lying between the vegetative hyphae.

Crystal violet staining. Note they contact several hyphae at the same
time. Scale bar, 20 m.

Fig. 13. A young fruit body of Psilocybe cubensis with a cap size of
8 mm. Note the already pigmented pileus with wet and slimy
appearance and the scarcity of whitish remains of the universal veil.
Fresh material. Scale bar, 2 mm.

Fig. 12. A detailed view from another location. Crystal violet

staining. Scale bar, 12 m.

appeared during an observation period of 6 wk. The onset of

the primordia was a relatively late phenomenon and occurred
in casing soil in which bacteria like Pseudomonas spp. (Fig. 23)
were present as natural inhabitants. Thick mycelial cords were
then formed above the interface between the compost and the
casing layer. Their growth direction was mainly vertical
towards the air-soil interface (Fig. 5). We detected the first
onset of the primordia as early as 4 d after the application of
casing soil alongside the mycelial cords. The primordia were
covered by fluffy white hyphae (FWH) like the mycelial cords
from which they had initiated.
LM, TEM and LTSEM revealed an abundance of needle-like
crystals on the surface of the hyphae growing vegetatively in
the casing soil (Figs 24, 44). However, no such crystals were
found on the hyphae beyond the enveloping zone (Figs. 25, Fig. 14. A vertical, median cut surface of the stipe of the same fruit
26, 45). Histologically, the cord tissue appeared as a closely body showing the beginning of the cavitation due to deletion of the
packed bundle of aggregating hyphae. The cord was enveloped whitish hyphae of the central core tissue. Scale bar, 1 mm.
by a relatively large zone of FWH containing crystals on their
surface and large air spaces between loosely arranged threads. sky-blue stained, round to oval globules were observed
Fig. 7 illustrates a newly formed mycelial cord with strongly between the FWH (Fig. 8) enveloping the cord tissue. We call
stained hyphal cells due to abundance of glycogen and them blue bodies. Most of the blue bodies (BB) were found
ribosomes in the cytoplasm (Figs 26, 30, 32). Many amorphous, between and in contact with hyphae (Fig. 9) as if binding them
M. Halit Umar and Leo J. L. D. Van Griensven 723

Fig. 17. A portion of the cap of Stropharia rugoso-annulata showing

white pyramidal structures of the universal veil as isolated and
shrunk remains. Fresh material. Cap size 45 mm. Scale bar, 6 mm.

Fig. 15. A vertical median cut surface of a 10 mm tall fruit body of

Coprinus domesticus. Note the absence of the cavitation of the stipe
at this growth stage. Fixed material. Scale bar, 2 mm.

Fig. 18. A vertical, median cut surface of the ascocarp of Peziza

ostracoderma. This almost transparent fruit body shows at least one
small and slime filled cavity. Fresh material. Scale bar, 3 mm.

Fig. 19. A median longitudinal cut surface of a young, 15 mm tall

Fig. 16. A portion of the cap of Coprinus sp. with a greyish tint of
ascocarp of Otidea onotica. Note the conical cavity under the cup.
the pileus with a maximal cap diam. of 12 mm. Note bright white,
Cytologically, numerous, severely deteriorating cells and countless
pyramidal shaped and loosely interconnected remains of the universal
fragments and granular cell remnants were found in this cavity. Fresh
veil which already disappeared in a part. Fresh material. Scale bar,
material. Scale bar, 4 mm.
5 mm.

together. Contrary to a bright yellow fluorescence of the frequently noticed the presence of remains of cells in the BB
hyphal filaments after acridine orange staining, BB remained (Figs 11, 12).
very weakly fluorescent with an orange to a greenish tint The BB were only found in the envelope of FWH around
unless they had enclosed parts of hyphae (Fig. 10). In LM we the developing mycelial cords. Ultrastructural studies of the
Morphogenesis and programmed cell death in fungi
Fig. 20. This fruit body of Volvariella bombycina is only 10 mm tall
and still grows within the closed volva. Note the slimy, pigmented
pileipellis and very conspicuous space between the cap and the volva.
Alcohol-preserved material. Scale bar, 1 mm.
hyphae within and around the BB showed significant cellular
changes : bleb formation by locally dilated nuclear membranes
(Fig. 29), an increase of the nuclear size with obviously
Fig. 21. Hyphae of Agaricus bisporus growing towards the centre of the rye grain. Arrows, chitosome. Scale bar, 2 m.
724
widened nuclear pores (Fig. 30), slight chromatin condensation
but obvious enlargement of the nucleoli (Fig. 31), intermingling
of the cytoplasm with karyoplasm, disappearance of the nuclei
with a coinciding hydropic degeneration (abnormal increase of
water in the cytoplasm) and cytoplasmic breakdown, an
apparent decrease of the stainability and osmiophilic charac-
teristic of the cell wall due to structural alteration, large masses
of degenerated cytoplasmic organelles, especially vesicles, and
thread-like cell fragments, between the hyphae (Figs 3034).
Inside the BB we found enlarged, empty or severely
degenerating hyphal cells, all of which showing ultrastructural
features indicating cell death (Figs 3539). In addition, for the
first time a slightly electron-dense, sometimes finely fibrillar
but amorphous matrix material appeared between the dead or
dying hyphal cells enclosed in the BB (Fig. 40). When the
usual PAS staining technique was applied to the non-
contrasted ultrathin sections containing the BB, we observed
diffuse, very fine, electron-dense granules both inside and
outside the cells resulting from a chemical reaction between
the free aldehyde molecules and leucofuchsin (Fig. 41). PAS
staining after a pretreatment with diastase revealed numerous
granules, almost exclusively extracellular. Inside the cells we
M. Halit Umar and Leo J. L. D. Van Griensven
Fig. 22. A hypha of Agaricus bisporus penetrates into the cellulosic
wall of the straw cell. Note the delicate and fibrillar interaction
between hypha and the wall. Scale bar, 02 m.
Fig. 23. A portion of the transitional zone of a fully developed
mycelial cord of A. bisporus in the casing soil containing a small
colony of bacteria nestled in the interhyphal mucilaginous substance.
Note the intimate contact between bacteria and hyphal walls. Scale
bar, 1 m.
725
have seen only a few reaction sites and, with difficulty,
recognised parts of the cytoplasmic membranes (Fig. 42).
These results indicated the presence of an extracellular
mucilaginous substance around cells.
LTSEM studies of the mycelial cord showed the distinct
anatomical zones : a large central zone of aggregated hyphae,
an outer (mantle) zone consisting of loosely arranged FWH,
and a narrow transitional zone coated with a layer of granular
and}or fibrillar material. Detailed observations ascertained the
presence of the ECM material, which connected the hyphae
together without completely obstructing the interhyphal
spaces, providing room for the accumulating gases (Fig. 45).
Results of comparative studies
The mycelial cord and primordia of Stropharia rugoso-annulata
(Fig. 6) showed features comparable with those found in A.
bisporus including abundant needle-like crystals on the FWH
surrounding the cord and primordia, and the BB formation by
degrading cells. Chalk white conical remnants of the universal
veil were observed on the caps of newly formed fruit bodies
(Fig. 17) as it was the case in Coprinus sp. (Fig. 16). When they
continued to grow the pigmented pileipellis turned wet and
slimy due to elaboration of a mucoid zone, and the conical
remnants rapidly decreased in size and in number.
Similarly, the cap surface of Psilocybe cubensis became
increasingly smooth, shiny and slimy (Fig. 13) as the fruit
bodies continued to grow. A mucoid zone of the pileipellis
was detected throughout entire developmental stages. This
pileal mucoid zone was even observed in the smallest
specimens of primordia with a cap size of 23 mm (Fig. 46).
LM studies revealed numerous deteriorating hyphal cells
spread in a wide and amorphous ground substance of the
pileipellis (Figs 47, 48). Most of the hyphae were severely
fragmented and the cells were apparently degrading. TEM
showed ultrastructural features comparable to those of cell
death seen during the histogenesis of the mycelial cord of A.
bisporus (Figs 4954). Fruit bodies of Panaeolus cyanescens
displayed a similar mucoid zone of the pileipellis containing an
amorphous ground substance in which deteriorating cells
were embedded (Figs 55, 56). The central core of the stipe of
young fruit bodies of Coprinus domesticus turned into a
cylindrical cavity when they grew longer than 10 mm (Fig.
15). In this lumen many dead or degenerating hyphal cells and
a mucoid fluid were found as observed in the stipe cavitation
of Psilocybe cubensis (Fig. 14) and Panaeolus cyanescens. Their
pileipellis also became wet and slimy, and showed a mucoid
substance especially between the deteriorating hyphae of the
outer layer beneath the conical remains of the universal veil.
Similarly, the pileipellis of growing Psathyrella candolleana
steadily became mucoid, and the stipe acquired a lumen in
which remains of dead cells were found. Mucoid pools of
microscopical size were detected in Peziza ostracoderma with a
cup size from 2 mm on (Fig. 18). These pools contained dying
segments of hyphae. In Otidea onotica the young fruit bodies
growing beyond 8 mm always showed a cone-shaped cavity
in an upside down position bordering on the developing cup
(Fig. 19). Cytologically, a semi-viscous mucoid fluid containing
many dead cells or severely degraded cells and an excessive
Morphogenesis and programmed cell death in fungi
Fig. 24. A portion of FWH showing negative-stained crystals emerging from the wall. Note large glycogen rosettes and other cell
organelles. Scale bar, 05 m.
Fig. 25. Hyphae of the transitional zone of a newly formed mycelial
cord and large interhyphal spaces (*) filled with fine fibrillar
mucilaginous substance. One of the hyphae with empty cytoplasm
apparently involved in the process of morphogenetic cell death, and
possess a totally occluded dolipore (thick arrow). A regular dolipore
septum with its parenthesome indicated by thin arrows. Scale bar,
2 m.
726
amount of their granular remains were found inside the
cavities. The growing primordia of Volvariella bombycina (Fig.
20) were covered by a light-brown pigmented and mucoid
pileipellis in which many degrading cells were found. Also the
inner side of the volva showed similar aspects. Tissue sections
of Tremella mesenterica revealed an enormous amount of a
mucoid substance forming a matrix surrounding all hyphae
including exobasidia. In this ECM many deteriorating hyphal
cells were visible.
DISCUSSION
In this study we primarily aimed at investigating histogenesis
of the mycelial cord of A. bisporus. Our results showed the
occurrence of morphogenetic cell death during mycelial cord
formation. A similar phenomenon found in developing
primordia of A. bisporus has been reported (Umar & Van
Griensven, 1997 b). The repetitive occurrence of cell death in
relation to morphogenesis of a fungus drew our attention and
we searched for comparable situations in other species of
macrofungi.
Commercially available mushroom compost has its own
ecosystem of micro-organisms like actinomycetes (Van den
Bogart et al., 1993) and thermophilic fungi (Straatsma et al.,
1991). The cultivation process starts with the inoculation of
M. Halit Umar and Leo J. L. D. Van Griensven
26
27
28
Figs 2628. TEM features of a mycelial cord connecting to a
normally growing fruit body of A. bisporus which has a cap size of
4 cm. (*), the interhyphal space ; v, vacuole ; n, nucleus ; d, dolipore
septum ; p, parenthesome ; c, rhomboidal bio-crystal (inclusion body).
Fig. 26. Normal mycelial cord tissue with very closely interwoven
hyphae containing abundant cytoplasm and other cell organelles. A
normal dolipore septum is arrowed. Scale bar, 3 m. Fig. 27. The
dolipore septum and parenthesome clearly show disorganisation.
Both hyphal segments seem normal. Scale bar, 1 m. Fig. 28.
Protoplasmic streaming is visible through the dolipore septum. Note
three rhomboidal bio-crystals in different developmental stages, and
also mucilaginous material in the interhyphal space. Scale bar, 1 m.
this compost with spawn grains. Under the right climatic
conditions the compost becomes diffusely colonized by the
mycelium within 2 wk. Mycelial growth of A. bisporus on
compost is stimulated by the presence of the thermophilic
727
Fig. 29. Small parts of adjacent hyphae. Three nuclei with blebs of
the nuclear membrane. Note the slight condensation of the
chromosomes. Compare the features of the cytoplasm with Figs 21,
2326. Scale bar, 1 m.
Scytalidium thermophilum. The onset of the primordia is a
relatively late phenomenon and takes place after the
application of a layer of the casing soil which consists of peat
and calcium carbonate or sugar mill waste, and contains micro-
organisms such as Pseudomonas spp. as natural inhabitants
(Danell, Alstro$ m & Temstro$ m, 1993) playing a possible
promoting role for the formation of mycelial cords and
primordial initiation (Ingratta & Patrick, 1986). The fruit
bodies are attached to a huge mycelial mass, which is
exploiting food resources in the compost through a network
of mycelial cords (Agerer, 1992) from which they initiate.
Mycelial cord formation precedes the onset of primordia.
During this study we have observed that no primordia were
generated if mycelial cords were not formed. Spawning of the
compost provides a substantial amount of mycelium with
known biological characteristics for cultivation of a particular
species and strain of mushrooms. Microscopic studies
demonstrated that hyphae on the spawn grains readily
penetrated the layers of the husk and spread within the
endosperm through the aleurone cell layer (Figs 13).
Cytological smear preparations showed that hyphae on the
surface of spawn grains were covered with many crystals of
Morphogenesis and programmed cell death in fungi 728

30 31

32 33 34
Figs 3034. Nuclear, cellular, and extracellular changes occurring during the development of the blue bodies. Fig. 30. Nuclear pores
(arrows) are widened and the nuclear envelope is ruptured. No chromatin condensation is noticeable. Except for swollen mitochondria
and slightly dilated endoplasmic reticulum all other cell organelles are normal. Scale bar, 06 m. Fig. 31. Nucleus with widened nuclear
pores and with a large nucleolus. The karyoplasm is continuous with the cytoplasm which becomes loose and hydropically degenerated.
Note the partial disappearance of the cell membrane and lytic changes of the cell wall (w). Scale bar, 04 m. Fig. 32. Large spaces
between normal looking hyphae are filled with many fragmented cell organelles (*). Scale bar, 25 m. Fig. 33. Severe hydropic
degeneration, cytoplasmic dissolution, and irregular and lytic cellular remains are visible. Scale bar, 06 m. Fig. 34. Degenerated
remnants of the cell walls form bridges between the hyphae. Scale bar, 06 m.

calcium oxalate. In contrast, the hyphae penetrating within the
grains contained no crystals on their walls (Fig. 21). On the
walls of hyphae growing in the compost after spawning (Fig.
4) numerous oxalate crystals were also found, but when
penetrating into the cell walls of the straw the crystals
practically disappeared (Fig. 22). Most of the hyphae located
within the straw cells again acquired calcium oxalate crystals.
These observations indicate that the formation of crystals on
the wall surface is dependent on the micro-environment and
the interaction between biological materials and hyphae. We
demonstrated that the calcium oxalate crystals were also
found on the surface of FWH (Figs 24, 44) in contrast to the
tissue forming hyphae (Figs 23, 2528, 45). Anton de Bary,
who for the first time described the occurrence of oxalate
crystals associated with the hyphae of Agaricus campestris in
1887 (Whitney & Arnott, 1987), related the chalky white
appearance of the mycelium to the presence of crystals.
Whitney & Arnott (1987) proved the existence of the dense
covering of needle-like calcium oxalate crystals on the hyphae
grown on rye grain and also in the aerial hyphae of A. bisporus
grown in agar culture. They determined that the crystals were
initially formed within the hyphal wall. Our microscopical
observations support their view (Fig. 24). They also thought
the hydrophobic characteristics of the aerial hyphae, which
function as a defence mechanism against bacterial and fungal
infections, are enhanced by the deposition of calcium oxalate
crystals on their surface. Furthermore, oxalic acid secreted into
the micro-environment around the hyphae has the potential of
binding calcium ions, which are abundantly present in casing
soil. Such chemical reactions may direct or alter the signal
transmission and intercellular communication necessary for
the histogenesis. Masaphy et al. (1987) reported the occurrence
of numerous oxalate crystals on the vegetatively growing
hyphae of A. bisporus. They found no such crystals, however,
on the hyphae of microscopic or more developed primordia.
Their observations confirmed findings of Eger & Su$ cker
(1964). In addition, they noticed that the interaction between
the hyphae and rod-like bacteria in casing soil caused the
disappearance of the crystals and helped in strand formation.
Our observations (Fig. 23) agree with their results ; we also
think that the role of bacteria in fungal histogenesis and in the
initiation of the primordia is indirect. Actively exploring
M. Halit Umar and Leo J. L. D. Van Griensven 729

hyphae during the penetration of the wall of straw cells, for

35
36
Figs 35, 36. TEM features of further developed blue bodies. In both
figures most of the hyphae are empty or severely degenerated. Due
to lytic changes and disintegration, cell walls look like blurred
remnants. Scale bars, 2 m.
example, contain no crystals (Fig. 22) as Wood et al. (1985)
found no calcium oxalate crystals on growing tips of hyphae.
All these reports and our results indicate the importance of the
presence or absence of calcium oxalate crystals on the hyphae
as a morphological sign in the assessment of their functional
and cellular differentiation during the growth processes.
We accept that the mycelial cord (Figs 5, 6) represents a
well-organized fungal tissue in contrast to the growth
characteristics of the FWH on the mushroom compost (Fig. 4).
In this cord tissue we find no crystals but an interhyphal
amorphous material, the ECM, binding the hyphae together
(Figs 25, 26, 45). This fact implicates a functional trans-
formation of the FWH towards organisation by means of the
production of the ECM. Vegetatively growing, widely
separated and overlapping hyphae are apparently not capable
of fulfilling this function of making fungal tissues by mere
elongation, branching and generating new cells. In this
investigation, we determined that an end comes to this solely
vegetative growth pattern as soon as the casing soil is applied
to the colonized mushroom compost, and fungal tissue
formation starts with morphogenetic cell death. The induction
of histogenesis by a process of cell death may seem
controversial but this premature event in a growing mycelium
presumably provides both the optimal conditions and the
matrix material needed for the initiation of the mycelial cord
tissue. The LM features of the newly formed mycelial cords of
microscopical size are illustrated in Figs 712. They show one
striking aspect, the occurrence of sky-blue coloured amorphous
globules varying in size between the FWH, the so-called blue
bodies. We have not found any other report describing such
structures around the developing cords. We observed the BB
mainly during mycelial growth in the casing soil. Their
number varies slightly under different conditions ; they are
more numerous and larger when the specimens were gathered

37 38 39
Figs 3739. Ultrastructure of the BB : hyphal remnants. Scale bars, 1 m. Fig. 37. A group of dead cells. Fig. 38 and Fig. 39 show
large groups of severely degraded, empty looking carcasses of dead hyphal cells.
Morphogenesis and programmed cell death in fungi
Fig. 40. A sharply delineated, fibrillar, osmiophilic matrix material appears between the hyphae of the transitional zone of the mycelial
cord as a binding substance. Scale bar, 1 m.
after irrigation, and they almost completely disappear at the
base of primordial initials, especially when they are growing
on the soil surface. The BB consist essentially of dead hyphal
cells (Figs 12, 3540) embedded in an abundant mucoid
substance as we demonstrated using PAS staining (Figs 41,
42). Both the time of their emergence during the growth
process and their selective location between the FWH strongly
suggest that they may have a specific function in the
histogenesis of the mycelial cord. We think that the BB
provide the primary ECM at the beginning of this process
(Figs 25, 40).
The matrix is an environment which is suitable for the
transmission of different kinds of morphogenetic signals in all
directions. Its abundance or scarcity depends on the tissue
type and also on the species. In jelly fungi, for example, a
gelatinous matrix constitutes the greatest part of the mass
while in A. bisporus and in many other fungi the ECM is
almost invisible with LM unless special histochemical
techniques like PAS stain are applied (Bullock & Willetts,
1996 ; Umar & Van Griensven, 1997 a). In our opinion, the
production of the primary ECM by the BB has a promoting
role for cellular communication, commitment and differ-
entiation by the creation of a new, radically changed
environment for the hitherto vegetatively growing hyphae ;
once a new gene expression is effected, the tissue forming
hyphae will then be capable of being self-sustaining in the
production of the ECM by functional specialization. It is
730
obvious that in the development of A. bisporus (i) the
morphogenetic cell death is an indispensable phenomenon,
and (ii) it happens more than once in the life span of a
primordium.
The occurrence of the BB does not depend on cell necrosis
caused by various kinds of external injurious factors, and all
hyphae around the BB continue to grow and show normal LM
and TEM features (Figs 11, 12, 3537, 40). The BB are rather
solitary globules but their distribution is obviously not
haphazard. Inside the blue bodies we almost exclusively find
dying cells and remnants of dead cells as severely degenerated
fragments embedded in a mucoid substance (Figs 32, 3740).
TEM studies indicate that first the nuclei are involved in the
process of the BB formation (Figs 2931). This is followed by
a series of structural changes of the cytoplasm and of the cell
wall, respectively (Figs 3239). These features are almost
similar to those found in morphogenetic cell death preceding
the development of hymenial chamber in primordia of A.
bisporus (Umar & Van Griensven, 1997 b).
The above mentioned observations and findings led us to
formulate the following hypothesis : (i) vegetatively growing
FWH containing calcium oxalate crystals on their surface
represent an undifferentiated state of growth ; (ii) an ECM is
needed for histogenesis ; (iii) the BB provide the primary ECM ;
(iv) once the hyphae form the minute mycelial cords, their
cells acquire the ability to produce the ECM and they lose
their crystals ; (v) mycelial cord is the first fungal tissue ; (vi) it
M. Halit Umar and Leo J. L. D. Van Griensven 731

43
41

42
44
Figs 41, 42. Application of the PAS staining on the non-contrasted
ultrathin sections. BB around the mycelial cord of A. bisporus. Fig. 41.
PAS staining indicates the sites of reaction between leucofuchsin and
free aldehyde molecules as fine black dots which are present in all cell
parts and also in the interhyphal spaces. Scale bar, 06 m. Fig. 42.
After a prolonged digestion by diastase, the same PAS reaction
specifically marks the mucinous molecules which are abundantly
present in the interhyphal space (*) but very sparse in the cytoplasm
in which, except for small portions of cell membrane, no other
organelles are recognisable. Scale bar, 04 m.

is likely that a type of programmed cell death plays a key role

in the formation of BB and, consequently, in the histogenesis
of the mycelial cord of A. bisporus.
The physiological mechanisms and pathological conditions
leading to cell death were and still are a matter of great
interest for scientists in various disciplines. Long before Kerr,
Wyllie & Currie (1972) published on the naturally occurring,
non-accidental form of cell death, called apoptosis, many
nineteenth century researchers had observed and studied cell
death (Clarke & Clarke, 1996). After identification of death
genes (Sulston & Horvitz, 1977) and the genetically controlled
nature of apoptosis was recognized the term programmed
cell death became widely accepted and the subject has been
a major topic of developmental biology and pathology.
Ameisen (1996) suggested a co-evolutionary role for PCD
within the branches of the phylogenetic tree and it is
impossible to exclude members of Kingdom Fungi from this
universal phenomenon. There is, however, still no consensus
on the occurrence of PCD in fungi. Moore (1996) argued on
this matter as follows : The hypothesis of programmed cell
death is attractive but there is not enough evidence for it. In
our opinion, we have demonstrated a substantial amount of
45
Figs 4345. LTSEM features of the mycelial cord of A. bisporus. Fig.
43. An oblique cut surface of the cord clearly shows the compact
organization of the cord tissue surrounded by loosely arranged fluffy
hyphae. T, the transitional zone. Scale bar, 100 m. Fig. 44. The
fluffy hyphae contain many needle-like crystals on their walls. The
others (*) behind these crystal-bearing hyphae belong to the
transitional zone. They are patchily covered by a fine granular
material. Scale bar, 8 m. Fig. 45. This high magnification image of
the mycelial cord demonstrates portions of four tissue-forming
hyphae bound to each other by an extracellular matrix (*). An
enclosed interhyphal air space (a) is also visible. Scale bar, 02 m.
facts which provides a firm basis for us to claim that PCD
does, indeed, occur in fungi.
We have noticed that morphogenetic cell death both in
developing primordia and during the histogenesis of the
mycelial cord is a short-lasting phenomenon that serves for
pattern formation and further development. It is an important
hallmark. We have collected some specimens of A. bisporus
Morphogenesis and programmed cell death in fungi
46
47
48
Figs 4648. LM of Psilocybe cubensis with a cap size of 3 mm. Fig.
46. Crystal violet staining. The mucoid pileipellis appears as a pale
zone under the remnants of the universal veil (arrow). Scale bar,
05 mm. Fig. 47. The mucoid pileipellis (*) has tiny and degraded
hyphal cells embedded in a large amorphous, bluish ground substance.
Scale bar, 50 m. Fig. 48. A similar region of the mucoid pileipellis
is shown in high magnification. Arrows indicate a group of
pathologically changed hyphal cells. Scale bar, 20 m.
about 1220 mm tall in which gills and gill cavity were not
developed. These structures begin normally to develop when
the fruit bodies reach a length of 6 mm (Umar & Van
Griensven, 1997 b). In those abnormal fruit bodies no
histological evidence was found of the expected morpho-
genetic cell death. If no BB are formed, the hyphal growth
continues in a vegetative form and no organisation of mycelial
cords takes place in A. bisporus. These observations suggest
that finely tuned control mechanisms are involved in the
timing of the occurrence of cell death during development of
macrofungi. Morphogenesis is a continuous process in space
and in time ; it has a time dimension as pointed out by Moore
(1996). Death genes of macrofungi, although they are not yet
732
49
50
51
Figs 4951. TEM of the mucoid pileipellis of Psilocybe cubensis. Early
signs of nuclear and cytoplasmic changes and the ECM. These
hyphae are embedded in a wide, finely fibrillar, osmiophilic ground
substance. Fig. 49. Note the fragmented cell walls are moving away
in this substance making a kind of halo around the cells. Scale bar,
2 m. Fig. 50. A detailed view. Scale bar, 1 m. Fig. 51. Note the
condensation of the chromosomes and bleb formation of the nuclear
envelope. Scale bar, 04 m.
identified with appropriate methods, may operate with the
highest accuracy in a time dimension to achieve their key role
in fungal morphogenesis. We argue that morphogenetic cell
death, wherever and whenever it occurs in developing fungi,
has a genetically programmed nature ; it thus represents a type
of PCD.
This is, of course, a theoretical claim based on the
observation of the histogenesis of the mycelial cord and
M. Halit Umar and Leo J. L. D. Van Griensven
52
Figs 5254. TEM of the mucoid pileipellis of Psilocybe cubensis. Morphological changes in a later phase of the development. Fig. 52. A
large scale of cell degradation, cell wall fragmentation and a granular, fibrillar matrix are visible. Scale bar, 2 m. Fig. 53. Severe
fragmentation of the cell walls and a wide matrix material are shown. Scale bar, 1 m. Fig. 54. Detailed view of a part of Fig. 52. Scale
bar, 1 m.
55 56
Figs 5556. TEM of the mucoid pileipellis of Panaeolus cyanescens.
Both figures show that the hyphae are encircled with and immersed
in a mucoid, fibrillar matrix. Scale bars for both figures, 2 m.
pattern formation during primordial development of A.
bisporus. It must be sustained with further observations and
with a variety of experimental methods. A series of findings
supports our claim : (i) the BB formation in the mantle zone of
FWH around the mycelial cord of Stropharia rugoso-annulata,
and many degrading cells and production of a mucilaginous
substance especially beneath whitish cones of their caps (Fig.
17) ; (ii) many severely degenerated and degrading cells in the
mucoid pileipellis of developing fruit bodies of both Psilocybe
cubensis (Figs 13, 4648) and Panaeolus cyanescens ; (iii) a
massive break-down of the central core hyphae of Coprinus
domesticus to make a cylindrical cavity in the stipe when they
grow taller than 10 mm, and similarly in Psathyrella candolleana
and in all other macrofungi studied (Fig. 14) ; (iv) degrading
cells in the mucoid pileipellis of the developing fruit bodies of
Volvariella bombycina within the volva (Fig. 20) ; (v) minute
733
53 54
slime-containing cavities in the fruit bodies of Peziza
ostracoderma (Fig. 18) ; (vi) massive cell degradation during
formation of a cavity under the ascus of Otidea onotica (Fig.
19) ; (vii) degrading or already dead cells in vast matrix of
Tremella mesenterica.
All structures like volva, annulus, remnants on the caps (Fig.
13, 16, 17, 20) and others, which are thought to be the
derivatives of the universal veil, are temporary constituents of
the developing macrofungi, and they regress even before the
fruit bodies become mature. The demise of such particular
parts, for example in bulboangiocarpic development (Moore,
1995) of V. bombycina, in the cavitation of the stipes, in the
production of a large quantity of slime in jelly fungi (Moore,
1965), in the formation of conical, slime-filled cavities in
ascomycetes, and the cellular changes characterising natural
senescence (Umar & Van Griensven, 1997 c) of A. bisporus can
possibly be explained by PCD. There are, however, both
similarities and differences between apoptosis in animals and
PCD in macrofungi : bleb formation of the nuclear membrane
(Fig. 29) instead of cell membrane, enlarged nuclear pores and
a considerable increase of the nuclear size (Figs 30, 31) were
the first objective signs for an unusual process. Condensation
of the chromatin was not obvious ; neither was the formation
of the so-called apoptotic bodies. The nuclei disappeared
without fragmentation, and cytoplasm showed dramatic
changes due to disorganisation and accumulation of excessive
amounts of water, rupture of vacuolar and vesicular membranes
and lysis of the organelles. The alteration of the composition,
thickness, and other structural characteristics of the chitinous
cell wall followed. While cells apparently enlarged in size,
cytoplasm shrunk to an almost negligible mass which gave the
cells the appearance of an empty carcass (Figs 3740) which
is a feature absolutely different from the protoplasmic
streaming through the dolipore septa from one segment to
another (Figs 27, 28). Parallel to these events the cell walls
became permeable and ruptured at several places providing an
Morphogenesis and programmed cell death in fungi
easy passage to the cytoplasmic elements to dislocate into the
interhyphal space. Degradation of cell remnants by extra-
cellular enzymes like chitinase is analogous to the phagocytosis
which occurs in animals, as we found no cell remnants in the
transitional zone of the mycelial cord but a large amount of
mucilaginous substance around the hyphae (Fig. 25). In the
histogenesis of mucoid pileipellis similar mechanisms of
enzymatic break-down of the cell walls may also create a
glucan-rich ground substance (Figs 4956).
In this investigation we showed that a type of mor-
phogenetic cell death differing from cell necrosis occurs
during the formation of the mycelial cord of A. bisporus as well
as during the histogenesis of the mucoid pileipellis of Psilocybe
cubensis. The well-timed occurrence and the predictable
morphological aspects indicate the importance of the phenom-
enon cell death for the morphogenesis in fungi. The
development and the final body plan of the fungi are thus not
secured only by hyphal growth and branching, and pro-
liferating cells but also by an active process of selective cell
deletion as seen in the development of animal embryos
(Coucouvanis & Martin, 1995 ; Mori et al., 1995). PCD
provides an elegant solution to developing organisms in
pattern formation. The role of PCD has now been recognised
and studied in the development of plants (Greenberg, 1996 ;
He, Morgan & Drew, 1996), in the nematode Caenorhabditis
elegans (Yuan, 1996), in prokaryotic organisms (Yarmolinsky,
1995 ; Chaloupka & Vinter, 1996), in unicellular eukaryotes
(Ameisen, 1995), in cellular slime mould Dictyostelium
discoideum (Cornillion et al., 1994) and in the yeast Schizo-
saccharomyces pombe (Torgler et al., 1997). These reports
support the view of the universal occurrence of the PCD
(Ameisen, 1996).
In fungi, the mechanisms involved in cavitation, which is an
essential landmark (Moore, 1996), could also be explained by
cell death with the coexistence of mechanical forces emerging
as a consequence of continuing growth. Lu (1991) reported
the role of cell degeneration in the gill formation during the
development of Coprinus cinereus. Moore (1965) investigated
fungal gel tissues in discomycete subfamilies and in the
Tremellales, and came to the conclusion that the gel was
produced by hyphal disintegration as well as by secretion.
Tissues of naturally ageing fruit bodies revealed cytological
features of cell death, which were obviously different to those
of infected or necrotic cells (Umar & Van Griensven, 1997 c).
Although the striking morphological features of morpho-
genetic cell death determined in this investigation are not
completely comparable to those of apoptosis taking place in
animals (Duke, Ojcius & Young, 1996) and the molecular
mechanisms involved are not known yet, we think that we
have found enough arguments to propose a hypothesis with
respect to the occurrence of programmed cell death in
developing mycelium and macrofungi.
Suggestions on the subject and critical comments on the
manuscript by Dr D. Moore are very much appreciated. We
also thank our colleagues Drs A. S. M. Sonnenberg,
G. Straatsma, F. P. Geels and J. P. G. Gerrits for their valuable
criticisms.
734
REFERENCES
Agerer, R. J. (1992). Ectomycorrhizal Rhizomorphs : Organs of Contact. In
Mycorrhizas in Ecosystems. 3rd European Symposium on Mycorrhizas,
Sheffield, England, 1991 (ed. D. J. Read, D. H. Lewis, A. H. Fitter &
I. J. Alexander), pp. 8490. C.A.B. International : Wallingford, U.K.
Ameisen, J. C. (1996). The origin of programmed cell death. Science 272,
12761279.
Ameisen, J. C., Idziorek, T., Billautmulot, O., Loyens, M., Tissier, J. P.,
Potentier, A. & Quaissi, A. (1995). Apoptosis in a unicellular eukaryote
(Trypanosoma cruzi) Implications for the evolutionary origin and role of
programmed cell death in the control of cell proliferation and survival. Cell
Death & Differentiation 2, 285300.
Bullock, S. & Willetts, H. J. (1996). Ultrastructural and histochemical studies
on mycelial germination of sclerotia of Sclerotinia minor. Mycological
Research 100, 561570.
Carlile, M. J. (1995). The success of the hypha and mycelium. In The Growing
Fungus. (ed. N. A. R. Gow & G. M. Gadd), pp. 319. Chapman & Hill :
London, U.K.
Chaloupka, J. & Vinter, V. (1996). Programmed cell death in bacteria. Folia
Microbiologica 41, 451464.
Clarke, P. G. H. & Clarke, S. (1996). Nineteenth century of research on
naturally occurring cell death and related phenomena. Anatomy &
Embryology 193, 8199.
Cornillon, S., Foa, C., Davoust, J., Buonavista, N., Gross, J. D. & Goldstein, P.
(1994). Programmed cell death in Dictyostelium. Journal of Cell Science 107,
26912704.
Coucouvanis, E. & Martin, G. R. (1995). Signals for death and survival : A
two-step mechanism for cavitation in the vertebrate embryo. Cell 83,
279287.
Danell, E., Alsto$ rm, S. & Temstro$ m, A. (1993). Pseudomonas fluorescens in
association with the fruit bodies of the ectomycorrhizal mushroom
Cantharellus cibarius. Mycological Research 97, 11481152.
Duke, R. C., Ojcius, D. M. & Young, J. D.-E. (1996). Cell suicide in health and
disease. Scientific American 275 (6), 4855.
Eger, G. & Su$ cker, I. (1964). Besteht ein Zusammenhang zwichen
Oxalatausscheidung und Fruchto$ rperbildung beim Kulturchampignon
Agaricus bisporus (Lge) Sing. ? Archiv fuX r Mikrobiologie 49, 275282.
Greenberg, J. T. (1996). Programmed cell death : A way of life for plants.
Proceedings of the National Academy of Sciences U.S.A. 93, 1209412097.
He, C., Morgan, P. W. & Drew, M. C. (1996). Transduction of an ethylene
signal is required for cell death and lysis in the root cortex of maize during
aerenchyma formation induced by hypoxia. Plant Physiology 112, 463472.
Ingratta, F. J. & Patrick, Z. A. (1986). Influence of microorganisms and
fungistasis on sporophore initiation in A. brunnescens. In Proceedings of the
International Symposium of Scientific & Technical Aspects of Cultivating Edible
Fungi (ed. P. J. Wuest, D. J. Royce & R. B. Beelman), pp. 2740. Elsevier :
Amsterdam.
Kerr, J. F. R., Wyllie, A. H. & Currie, A. R. (1972). Apoptosis : a basic
biological phenomenon with wide-ranging implications in tissue kinetics.
British Journal of Cancer 26, 239257.
Lillie, R. D. & Fullmer, H. M. (1976). Polysaccharides ; Mucins. In Histo-
pathologic Technic and Practical Histopathology (ed. R. D. Lillie &
H. M. Fullmer), pp. 611678. McGraw-Hill Book Company, A. Blakiston
Publication : New York.
Lu, B. C. (1991). Cell degeneration and gill remodelling during basidiocarp
development in the fungus Coprinus cinereus. Canadian Journal of Botany 69,
11611169.
Masaphy, S., Levanon, D., Tchelet, R. & Henis, Y. (1987). Scanning electron
microscopic studies of the interaction between Agaricus bisporus (Lange)
Sing. hyphae and bacteria in casing soil. Applied & Environmental
Microbiology 53, 11321137.
Moore, D. (1995). Tissue formation. In The Growing Fungus (ed. N. A. R. Gow
& G. M. Gadd), pp. 423465. Chapman & Hill : London, U.K.
Moore, D. (1996). Inside the developing mushroom-cells, tissues and tissue
patterns. In Patterns in Fungal Development (ed. S. W. Chiu & D. Moore), pp.
136. Cambridge University Press : Cambridge, U.K.
Moore, E. J. (1965). Fungal gel tissue ontogenesis. American Journal of Botany
52, 389395.
M. Halit Umar and Leo J. L. D. Van Griensven
Mori, C., Nakamura, N., Kimura, S., Irie, H., Takigawa, T. & Shiota, K. (1995).
Programmed cell death in the interdigital tissue of the fetal mouse
limb is apoptosis with DNA fragmentation. Anatomical Record 242,
103110.
Straatsma, G., Gerrits, J. P. G., Gerrits, Th. M., Op den Camp, H. J. M. & Van
Griensven, L. J. L. D. (1991). Growth kinetics of Agaricus bisporus mycelium
on solid substrate (mushroom compost). Journal of General Microbiology
137, 14711477.
Sulston, J. E. & Horvitz, H. R. (1977). Post-embryonic cell lineages of the
nematode Caenorhabditis elegans. Developmental Biology 56, 110156.
Torgler, C. N., Detiani, M., Raven, T., Aubry, J. P., Brown, R. & Meldrum, E.
(1997). Expression of BAK in Schizosaccharomyces pombe results in a lethality
mediated through interaction with the calnexin homologue CNX1. Cell
Death & Differentiation 4, 263271.
Umar, M. H. & Van Griensven, L. J. L. D. (1997 a). Hyphal regeneration and
histogenesis in Agaricus bisporus. Mycological Research 101, 10251032.
Umar, M. H. & Van Griensven, L. J. L. D. (1997 b). Morphogenetic cell death
in developing primordia of Agaricus bisporus. Mycologia 89, 274277.
Umar, M. H. & Van Griensven, L. J. L. D. (1997 c). Morphological studies on
735
the life span, developmental stages, senescence and death of the fruit
bodies of Agaricus bisporus. Mycological Research 101, 14091422.
Van den Bogart, H. G. G., Van den Ende, G., Van Loon, P. C. C. & Van
Griensven, L. J. L. D. (1993). Mushroom workers lung : serologic reactions
to thermophilic actinomycetes present in the air of compost tunnels.
Mycopathologia 122, 2128.
Van Gils, J. J. (1988). Cultivation. In The Cultivation of Mushrooms (ed.
L. J. L. D. Van Griensven), pp. 263308. Darlington Mushroom Labora-
tories Ltd : Rustington, U.K.
Yarmolinsky, M. B. (1995). Programmed cell death in bacterial population.
Science 267, 836837.
Yuan, J. Y. (1996). Evolutionary conservation of a genetic pathway of
programmed cell death. Journal of Cellular Biochemistry 60, 411.
Whitney, K. D. & Arnott, H. J. (1987). Calcium oxalate crystal morphology
and development in Agaricus bisporus. Mycologia 79, 180187.
Wood, D. A., Craig, G. D., Atkey, P. T., Newsam, R. J. & Gull, K. (1985).
Ultrastructural studies on the cultivation process and growth and
development of the cultivated mushroom Agaricus bisporus. Food Micro-
structure 4, 143164.