10.6092/issn.2531-7342/16112
The ascomycetes in the genus Morchella (commonly called morels) have a considerable economic and biotechnological value for their culinary and medicinal proprieties. However, their biotechnological interest is not limited to the fruiting body cultivation, but also in mycelial production to obtain bioactive compounds and other biotech products. In order to better exploit the biotechnological potentialities of morels it is necessary to improve the knowledge on their biology and mycelial characteristics. In this paper morphological and growth characteristics of mycelia, as well as biomass formation of Italian collections of five edible medicinal Morchella species [M. esculenta (L.) Pers., M. dunalii Boud., M. importuna M. Kuo, O'Donnell &amp; T.J. Volk, M. disparilis Loizides &amp; P.-A. Moreau and M. purpurascens (Krombh. ex Boud.) Jacquet.] are presented.
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Zhao Q, Lv M, Li L, Huang W, Zhang Y, Hao Z (2021) "Temptation" and "Trap" of Morchella industry in China. Journal of Yeast and Fungal Research 19(4):232–237. https://doi.org/10.13341/j.jfr.2021.1447
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                IJM - Italian Journal of Mycology
                ISSN 2531-7342 - Vol. 52 (2023): 50-61
                Journal homepage: https://italianmycology.unibo.it/

Research article

              Morphological observation and biomass formation
              in different edible medicinal Morchella collections
                         (Pezizomycetes, Ascomycota)

     Susanna M. Badalyan1, Narine Gharibyan1, Carmelo Gianchino2, Mirco Iotti2,
                             Alessandra Zambonelli3
1
  Laboratory of Fungal Biology and Biotechnology, Institute of Biology, Yerevan State University, Yerevan, Armenia
2
  Department of Life, Health and Environmental Science, University of L’Aquila, via Vetoio, 67100, Coppito, L’Aquila,
Italy
3
  Dipartimento di Scienze e Tecnologie Agro-Alimentari, University of Bologna, Bologna, Italy

Corresponding author e-mail: s.badalyan@ysu.am


Article info
Received 30/12/2022; accepted 20/2/2023
https://doi.otg/10.6092/issn.2531-7342/16112

Abstract
The ascomycetes in the genus Morchella (commonly called morels) have a considerable economic and
biotechnological value for their culinary and medicinal proprieties. However, their biotechnological interest is
not limited to the fruiting body cultivation, but also in mycelial production to obtain bioactive compounds and
other biotech products. In order to better exploit the biotechnological potentialities of morels it is necessary
to improve the knowledge on their biology and mycelial characteristics. In this paper morphological and
growth characteristics of mycelia, as well as biomass formation of Italian collections of five edible medicinal
Morchella species [M. esculenta (L.) Pers., M. dunalii Boud., M. importuna M. Kuo, O’Donnell & T.J. Volk,
M. disparilis Loizides & P.-A. Moreau and M. purpurascens (Krombh. ex Boud.) Jacquet.] are presented.

Keywords
Morels, mycelia, morphology, biomass, Morchella esculenta, Morchella dunalii, Morchella importuna,
Morchella disparilis, Morchella purpurascens


Introduction

Morchella species, the true morels (Morchellaceae, Pezizales and Pezizomycetes) belong of the
phylum Ascomycota (Hibbett et al., 2007). They are mostly distributed in the northern hemisphere
and develop ascomata for only a few weeks in early spring. Taxonomy, phylogeny and biogeography,
ecological diversity and distribution, genomics and cultivation of Morchella spp. have been reviewed
(Barseghyan et al., 2012; Du et al., 2015). Based on ascoma morphology, these macrofungi were
placed into three groups: black, yellow and semi-free capped morels. However, according to recent
molecular studies at least 80 species were recognized, mostly from China (Du et al., 2019; Loizides et
al., 2021). Most of them are saprobic but some species seems to establish complex interactions with the
roots of the plants which are far to be completely clarify (Ori et al., 2019). Morels have considerable
economic and biotechnological values for their culinary and medicinal proprieties (Badalyan, 2012;
Tietel and Masaphy, 2018; Badalyan and Zambonelli, 2019).

Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and maintained by ABIS-AlmaDL. © 2023 The Authors. This work is licensed under CC BY 4.0
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    Wild edible morels are also harvested and commercially exported from China, India, Turkey,
Mexico, and the United States to European countries, such as France, Belgium and Switzerland, as
fresh, frozen or dried organic food products (Pekşen and Akdeniz, 2012). The annual export of dried
morels in China increased fivefold from 181,000 kg to 900,000 kg over the past five years, with
average price 160 USD kg-1.
    True morels with their nutritional value, meaty texture, unique aroma and delicate flavor (bitter,
umami, sour, sweet, salty and mouth-drying) are among the highly prized edible mushrooms (Beluhan
and Ranogajec, 2011; Heleno et al., 2013; Vieira et al., 2016; Badalyan and Zambonelli, 2019).
Several volatile and aromatic compounds, such as 1-octen-3-ol, 1-octadecanol, cyclooctylalcohol,
silanediol, 2-methylaminoethanol, aldehydes, terpenoids, esters, phenolics, ketones and others have
been detected in Morchella spp. (Taşkın, 2013; Tietel and Masaphy, 2018; Sunil and Xu, 2022).
    Due to delicious flavour and short fruiting season, the worldwide demand for morels has stimulated
their cultivation industry. Saprobic Morchella species (Morchella sextelata M. Kuo, M. eximia Boud.,
M. exuberans Clowez, Hugh Sm. & S. Sm., M. importuna M. Kuo, O’Donnell & T.J. Volk, M. oweri
X.H. Du, M. rufobrunnea Guzmán & F. Tapia, M. tomentosa M. Kuo) are widely cultivated in China
(Peng et al., 2015; He et al., 2019; Zhao et al., 2021; Xu et al., 2022). Total world production of the
true morel Morchella esculenta (L.) Pers. is 150 tons dry weight which is about 1.5 million tons fresh
weight with average price from 200 to 750 USD kg-1.
    Morels have also been appreciated by humans for their medicinal values (Hobbs, 1995; Badalyan,
2012; Ajmal et al., 2015; Badalyan and Zambonelli, 2019; Sunil and Xu, 2022). They have been
used in traditional medicine in China, India, and Pakistan to treat excessive sputum, gastrointestinal
disorders, and other pathological conditions (Paul et al., 2018). Recent studies suggested that morels
possess antimicrobial, anti-inflammatory and antioxidant (Nitha et al., 2007; Jander-Shagug and
Masaphy, 2010; Fu et al., 2013; Dissanayake et al., 2021), hepatoprotective (Nitha et al., 2013),
antitumor and immunomodulatory (Liu et al., 2018), antiviral, anti-leishmanial (Peretz et al., 2018),
neuroprotective and neuritogenic (Xiong et al., 2016; 2017), purgative, emollient, tonic, wound-
healing (Sunil and Xu, 2022) and other medicinal properties.
    The health-promoting effects and bioactivity of morel mushrooms have been mainly attributed
to a wide range of bioactive molecules (polysaccharides, tocopherols, ascorbic acid and vitamin D,
minerals, carotenoids, flavonoids, triglycerides, free fatty acids, sterols, organic and phenolic acids)
present in their wild and cultivated ascomata, as well as mycelia (Heleno et al., 2013; Tietel and
Masaphy, 2017; Dissanayake et al., 2021).
    Morels, used as nutraceuticals and nutriceuticals (functional food) are mostly gathered from
the wild due to difficulties to cultivate them especially outside of China. The increasing demands
of morels worldwide the further progress in fundamental fungal biology research and innovative
cultivation technologies are required (Arkan and Güler, 1992; Güler and Arkan, 2000). Cultivation
of fermented mycelium will expand production of Morchella as functional food, food flavoring and
other biotech products (Bulam et al., 2018). However, the high level of morphological variability of
mycelial characteristics of morels during different developmental stages of the life cycle, including
asexual reproduction modes, creates several problems for large scale biotechnological exploitation of
these mushrooms (Yuan et al., 2021). Further studies of morphological characteristics and growth rate
of vegetative mycelia, ecology and genomics of morels need to be addressed to protect their natural
resources.
    In this paper, morphological and growth characteristics of mycelia, as well as biomass formation
of Italian collections of five edible medicinal Morchella species [M. esculenta, M. dunalii Boud., M.
importuna, M. disparilis Loizides & P.-A. Moreau, M. purpurascens (Krombh. ex Boud.) Jacquet.]
are presented.

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Material and methods

Fungal cultures
Ten strains of M. esculenta, M. dunalii, M. importuna, M. disparilis, and M. purpurascens with
different origin isolated in central-north Italy have been studies (Table 1). They were isolated on potato
dextrose agar 25 g L-1 (PDA, Difco) by a portion of the sterile tissue of the stalk or by ascospores.
Ascospores were obtained by pipetting 100 µL of sterile water with 100 ppm of streptomycin within
alveola containing mature asci. After one or two ten-fold dilutions, 100 µL of ascospore suspension
was spread into Petri dishes containing PDA added with 100 ppm of streptomycin. After 12-18 h, the
germinating ascospores were detected under a stereomicroscope and transferred into new PDA Petri
dishes by a sterile needle.
   The identity of each isolated strain was confirmed by amplifying and sequencing their ITS nrDNA
region using the primer pair ITS1f-ITS4 (Gardes and Bruns, 1993) following the protocol of Bonuso
et al. (2006). Purification and sequencing reactions were performed by Microsynth seqlab (Balgach,
Switzerland) using both forward (ITS5) and reverse (ITS5i) primers. The obtained sequences were
deposited in Genebank under the accession numbers (Table 1).

Table 1 - Metadata of Italian collections of Morchella spp. used in this study
 Species             Strain            Origin        Region                  Locality                    Accession
                                                                                                         number
 M. disparilis       MdiSU1-1          Spore         Sardinia                Domusnovas                  OQ116872
 M. dunalii          MduNU2-4          Spore         Sardinia                Porto Ferro                 OQ116874
                     MduGR3-4          Spore         Tuscany                 Castiglione della Pescaia   OQ116873
 M. esculenta        MesAQ2-C          Spore         Abruzzi                 L’Aquila                    OQ116875
                     MesAQ6–2          Context       Abruzzi                 Castel del Monte            OQ116876
                     MesFI2-3          Spore         Tuscany                 Palazzolo sul Senio         OQ116877
 M. importuna        MimBL1-1          Spore         Veneto                  Auronzo di cadore           OQ116878
                     MimMO1-1          Spore         Emilia-Romagna          Vignola                     OQ116879
 M. purpurascens     MpuAQ1            Context       Abruzzi                 Castel del Monte            OQ116880
                     MpuTN2            Spore         Trentino Alto Adige     Madonna di Campiglio        OQ116881


   After molecular identification, the isolated strains were morphologically screen on PDA and liquid
malt-extract (ME: 200 mL 7o malt-extract obtained from the beer factory, 800 mL distilled water, pH
6.0) media.

Macromorphological observation
Mycelial inocula (5 mm3) of each strain were transferred to the centre of PDA plates (Ø 90 mm,
three plates per isolate) and incubated at 23 oC in the dark, except daily checks. Morphological
characteristics of colonies (form, texture, mycelial density, pigmentation of aerial mycelia and agar,
etc.) were described according to Stalpers’s scales (Stalpers, 1978). After 10 days of incubation in
the dark, observation of cultures, including sclerotia development was continued under a day/night
regime during 21 days.

Growth rate
The daily growth rate (GR, mm d-1) of mycelia on PDA was measured during 3-4 days until it
completely covered the agar plates. It was calculated by the following formula: GR = R1-R0/T, where

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R is radius of colony, T is the incubation time. Average growth rates (GRavr) were calculated from daily
GR values of each measurement. Each strain was repeated three times with inocula from the same
mother cultures. Data from six measurements from each strain were expressed as mean ± standard
deviation (SD).

Micromorphological observation
For micromorphological studies, including development of asexual spores and vegetative sclerotia,
the strains were grown on sterile microscope cover slips (2 × 2 cm, 2 - 3 for each plate) placed onto
PDA plates next to growing mycelia (Badalyan et al., 2011). The plates were incubated at 23 °C
and from the 3rd day when mycelia were starting to overgrow the cover slips they were removed
for observation under a microscope (Omano OM157T, USA) equipped with digital camera (× 0.5,
OptixCam, Summit Series, 10.0MP, USA) connected to a computer software program OptixCam
OCView v. 7.1. Micromorphological studies were carried out without staining. Two magnifications,
× 300 and × 1,200, have been applied. The dishes and preparations made from the growing mycelia
were observed during 3-4 weeks.
   The Erlenmeyer flasks with 100 mL ME medium were inoculated by 10 plugs (5 mm3 each) and
incubated at 23 oC under static conditions during 10 and 21 days. The micro- and macromorphological
growth characteristics of mycelia have been described (Supplementary Fig. S1).

Biomass formation
After 10 and 21 days of growth in static culture, the biomass samples were separated from cultural
liquid by filter paper (FILTRAK, FN 18), washed three times by distilled water, dried at 50‒60 oC and
weighted. The weight of obtained dry biomass samples were recalculated in g L-1.
   The taxonomic significance of observed mycelial characteristics of studied collections, such as
morphological, GR value, development of sclerotia and other asexual structures, as well as formation
of biomass were evaluated. The cultures were incorporated into Culture Collection of the Laboratory
of Fungal Biology and Biotechnology, Yerevan State University, as well as the culture collection of
the L’Aquila University.

Results and Discussion

Morphological observation
Mycelial colonies of studied Morchella strains on PDA were at first pale-creamy, later beige, yellow-
rusty or cinnamon brown. The pigmentation was starting from the centre of colonies and tended
to increase during the cultivation times. Aerial mycelia were well-developed, flappy-cottony, later
woolly-felt. The density of colonies increased during the growth, advancing from the surface of the
agar towards the plate cover (Supplementary Fig. S2). Agar was light or dark brown, rusty-brown to
cinnamon-brown (Table 2).
   After 5‒7 days of growth, development of spherical, rusty-brown or beige sclerotia from highly
proliferating intertwined hyphae of thickened and pigmented outer layer of colonies have been
described in all species/strains, except M. purpurascens MpuAQ1 (Supplementary Fig. S2).




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Table 2 - Morphological observation of mycelia of Morchella collections on PDA during 21 days of growth at 23 oC.
Species             Strain        Macro- and micromorphological characteristics
M. disparilis       MdiSU1-1 Colony initially creamy, light brown in the centre, cobweb-woolly, with even edge,
                                  later rusty-brown, cinnamon-brown, woolly-felt with well-developed fluffy aerial
                                  mycelium, agar cinnamon-brown, large sclerotia light to dark rusty-brown, brown
                                  over the entire surface of agar.
                                  Hyphae well-septate, brownish, granulate by large fatty inclusions anastomosed,
                                  vertically, later double and triple branched, arthroconidia are present.
M. dunalii          MduNU2-4 Colony creamy, light brown in the centre, cobweb, later felt, edge even, agar from
                                  light to dark brown. Rare, small, whitish-creamy, later large, orcha-rusty sclerotia
                                  along the edge of the colony on dense mycelial clusters.
                                  Hyphae septate, anastomosed, rusty-brown, granulate by large fatty inclusions,
                                  vertically later triple branched, clusters of budding cells, octahedral and cubic
                                  crystals and arthroconidia present.
                    MduGR3–4 Colony whitish-creamy, later light brown in the centre, cobweb, felt, edge even, agar
                                  light brown, brown, rare sclerotia light rusty. Hyphae well-septate, anastomosed,
                                  hyaline, later brownish-cinnamon, granulate by large fatty inclusions, vertically,
                                  double and triple branched.
M. esculenta        MesAQ2-C Colony whitish-creamy, cobweb-woolly, edge even, mycelium and agar rusty-
                                  brown, numerous rusty-brown sclerotia at the edges of colony.
                                  Hyphae well-septate, granulate by large fatty inclusions, vertically branched, with
                                  large drops of exudate; sclerotia composed of budding cells with typical out-growing
                                  spiny hyphae, arthroconidia present.
                    MesAQ6-2 Colony whitish-creamy, light brown in the centre, rusty-brown, cobweb-woolly-felt,
                                  edge even, agar rusty-brown, small sclerotia rusty-brown at the edges of colony.
                                  Hyphae well-septate, brown, initially branched vertically, granulate by large fatty
                                  inclusions, anastomosed. Brown sclerotia formed of budding cells with typical out-
                                  growing spiny hyphae, arthroconidia present.
                    MesFI2-3      Colony whitish-creamy, rusty-brown, brown, cobweb, woolly-felt, edge even, agar
                                  rusty-brown, brown; small rusty-brown sclerotia at the edge, later in the centre of
                                  colony.
                                  Hyphae well-septate, granulate by large fatty inclusions, initially branched vertically,
                                  sclerotia formed of budding cells with typical out-growing spiny hyphae.
M. importuna        MimBL1-1 Colony milky-creamy, beige, cobweb-woolly-felt, edge even, agar beige, light
                                  reddish-brown, beige-rusty to dark brown sclerotia along the edge of the colony.
                                  Hyphae well-septate, intensively granulate by fatty inclusions, vertically and
                                  dichotomously branched, with large drops of exudate, dark brown sclerotia composed
                                  of oval to round-shaped cells.
                    MimMO1-1 Colony milky-creamy, beige, fluffy, later cobweb-woolly-felt, edge even, agar light
                                  beige, dark brown sclerotia over the entire surface of colony.
                                  Hyphae well-septate, initially vertically, later dichotomously branched, intensively
                                  granulate with large fatty inclusions, with drops of exudate, clusters of ochre-brown
                                  hyphae and sclerotia composed of oval to round-shaped budding cells.
M. purpurascens     MpuAQ1        Colony creamy, fluffy-felt, later brown, dark cinnamon-brown, woolly-felt, denser
                                  in the centre and light brown along even edges, agar brown, dark cinnamon-brown;
                                  sclerotia absent.
                                  Hyphae well-septate, hyaline, vertically branched, finely granulated, anastomosed;
                                  horn-like branched hyphae, octahedral and cuboid crystals, arthroconodia present,
                                  sclerotia not observed
                    MpuTN2        Colony whitish, light brown in the centre, felt, woolly-felt, edge even, agar light
                                  brown, yellowish-rusty sclerotia over the entire surface.
                                  Hyphae well-septate, hyaline, vertically branched, anastomosed, finely granulated,
                                  horn-like hyphae, octahedral and cuboid crystals, yellowish-rusty sclerotia present.



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Table 3 - Morphological observation of Morchella collections growing on ME medium during 21 days at 23oC.
Species             Strain        Macro- and micromorphological characteristics
M. disparilis       MdiSU1-1 Well-developed substrate and poorly developed beige, later dark beige aerial mycelia
                                  with dark beige or dark brown sclerotia.
                                  Hyphae well-septate, brownish, granulate, vertically, double and triple branched,
                                  anastomosed.
M. dunalii          MduNU2-4 Well-developed substrate mycelium, dark beige aerial mycelium, numerous brown
                                  sclerotia in aerial and substrate mycelia.
                                  Hyphae well-septate, brown, granulate, brown sclerotia composed of budding oval-
                                  cylindrical cells.
                    MduGR3–4 Well-developed substrate mycelium, small brown sclerotia in aerial and substrate
                                  mycelia.
                                  Hyphae well-septate, brown, granulate, sclerotia composed of budding cylindrical
                                  cells.
M. esculenta        MesAQ2-C Well-developed substrate and poorly developed aerial mycelia, large dark beige,
                                  yellowish sclerotia in surface and substrate mycelia.
                                  Hyphae aseptate or well-septate, granulate, vertically and dichotomously branched.
                    MesAQ6–2 Well-developed substrate and greyish-brownish aerial mycelia, dark beige sclerotia
                                  in surface, later substrate mycelia.
                                  Hyphae aseptate or well-septate, granulate, vertically and dichotomously branched.
                    MesFI2-3      Well-developed substrate and poorly developed aerial mycelia, large yellowish, dark
                                  beige, brwon sclerotia in aerial and substrate mycelia.
                                  Hyphae well-septate or aseptate, granulate, vertically and dichotomously branched,
                                  rare octahedral crystals in cultural liquid.
M. importuna        MimBL1-1 Well-developed substrate and poorly developed aerial mycelia, brown sclerotia in
                                  aerial and substrate mycelia.
                                  Hyphae mainly aseptate, intensively granulate, sclerotia composed of oval-round
                                  shaped budding cells.
                    MimMO1-1 Well-developed substrate and poorly developed aerial mycelia, brown sclerotia in
                                  surface and substrate mycelia.
                                  Hyphae well-septate, granulate, vertically and dichotomously branched with large
                                  drops of exudate, clusters of ochre-brown hyphae and sclerotia with chain of
                                  compact oval budding cells.
M. purpurascens     MpuAQ1        Poorly developed substrate mycelium, aerial mycelium fluffy, woolly-felt, greyish-
                                  beige-brown, sclerotia absent.
                                  Hyphae septate, hyaline, brownish, poorly branched.
                    MpuTN2        Well-developed substrate mycelium, beige sclerotia in surface and substrate mycelia.
                                  Hyphae mostly aseptate, hyaline, brownish, brown sclerotia composed of round- and
                                  oval-shaped budding cells, horn-like hyphae present.

   The sclerotia differed in size, abundance and colour. In M. esculenta, M. disparilis and M.
importuna cultures, they were smaller, rusty-brown, beige-rusty and dark brown, respectively, while
in M. dunalii larger in size and ochre-rusty (Supplementary Figs 3a‒c). The sclerotia in M. importuna,
M. disparilis, and M. esculenta were more abundant over the surface of the agar, while in M. dunalii
they were developed only at the edge of the plates. Small, yellow-rusty sclerotia over the surface
of colony were rarely observed in M. purpurascens MpuTN2 strain isolated from stalk of ascomata
(Table 2 and Supplementary Fig. S2).
   Budding (oval, elongated, rounded, and cylindrical) cells (Figs 1a‒e) and spiny hyphae out-
growing from the sclerotia (Fig. 1f) have been reported as characteristic feature in observed
Morchella strains. Elongated-cylindrical to oval budding cells or chlamydospores were described in
M. disparilis MdiSU1-1 (Fig. 1a), cylindrical to rounded-oval cells in M. dunalii MduNU2-4 (Fig.
1b,c), and rounded to ovoid cells in M. importuna MimBl1-1 (Figs 1d,e) strains. The spiny hyphae

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out-growing from the sclerotia and budding rounded cells were typical for M. esculenta strains (Fig.
1f). Development, pigmentation, structure and abundance of sclerotia are considered as morphological
traits in studied Morchella spp.




Fig. 1 - Micromorphology of studied Morchella cultures: (a) sclerotia and budding cells in MdiSU1-1, (b,c) MduNU2-4,
(d,e) MimBl1-1, (f) sclerotium with out-growing spiny hyphae and budding cells in MesFI2-3, (g,h) double and triple
hyphal branching in MdiSU1-1, (i‒k) hyphal granulation in MdiSU1-1, MesAQ2-C and MimMO1-1, (l) hyphal
anastomoses in MdiSU1-1, (m) droplets of exudate in MesAQ2-C, (n) crystals in MpuTN2, (o) arthroconidia in MdiSU1-1,
(p) MesAQ2-C, (q) MpuAQ1 on PDA, (r) budding hyphae and (s) sclerotia in liquid cultures of MpuTN2 and MesFI2-3,
respectively. Bars = 10 µm.


   Several stages during sclerotia development in M. esculenta and M. conica Pers. strains have been
described: growth and branching of hyphae of primary mycelia, interweaving of hyphae of secondary
mycelia to form compact masses and formation, growth and maturation of sclerotia (Arkan and Güler,
1992). According to authors the vegetative chlamydospores and asexual conidia were also developed
during sclerotia formation suggesting that Morchella fungi display diverse reproduction and survival
strategies under different environmental conditions. Moreover, morphological development of
conidia and nuclear distribution during their asexual reproduction are little known and deserved
further studies (Yuan et al., 2021).


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   Hyaline or brown, well-septate and anastomosed hyphae (Fig. 1l) were typical for all studied cultures
(Table 2). Perpendicular initial hyphal branching was observed in all collections, while dichotomous
and triple branching in M. disparilis (Figs 1g,h), M. dunalii and M. importuna strains. Extensively
or finely granulated by fatty inclusions anastomosed hyphae were characteristic feature for studied
Morchella collections (Figs 1i‒l). Drops of exudate were observed in mycelia of M. esculenta (Fig.
1m) while cuboid, cylindrical and octahedral crystals in M. dunalii and M. purpurascens (Fig. 1n)
cultures. Horn-like hyphae were typical for M. purpurascens strains (Table 2 and 3). In studied strains
asexual arthroconidia were described in M. esculenta (MesAQ2-C and MesAQ6-2), M. disparilis
(MdiSU1-1), M. dunalii (MduNU2-4) and M. purpurascens (MpuAQ1) strains (Figs 1o‒q).
   Many authors referred asexual stage in the life cycle of several Morchella spp. (M. angusticeps
Peck, M. conica, M. semilibera DC., M. spongiola Boud., M. steppicola Zerova, M. esculenta) to
Costantinella-like morphotype (Paden, 1972; Volk and Leonard, 1990; Buchalo et al., 2009; Carris
et al., 2015). Costantinella is a genus of anamorphic fungi, such as Costantinella terrestris (Link)
S. Hughes, Costantinella cristata Matr., Costantinella clavata Matr. etc. Costantinella spp. which
are growing on soil, moss and woody debris. Phylogenetic linkage between both Morchella and
Costantinella morphotypes was confirmed by several cultural and molecular studies (Volk and
Leonard, 1990; Carris et al., 2015). Costantinella was also described in the related genera, such as
Disciotis Boud., Gyromitra Fr. and Hydnotrya Berk. & Broome (Carris et al., 2015). The widespread
occurrence of anamorphic morphotypes in morels suggests that the life cycles of these pleomorphic
and polymorphic fungi are more complex than previously recognized (Carris et al., 2015). Since
formation of mitospores in the life cycle of morels is well documented in culture, their role under
natural conditions is not clearly defined. However, their wide geographic occurrence suggests that
asexual conidia could play a role as fertilizing agents (Carris et al., 2015).
   The substrate mycelium during static (surface) growth was well-developed in all strains, except
M. esculenta MesAQ6-2 and M. purpurescens MpuAQ1 in which the aerial mycelia were developed
(Supplementary Fig. S1, Table 3). Budding and intensively granulated hyphae, beige, dark beige or
brown sclerotia were observed in aerial and substrate mycelia of all strains, except M. purpurescens
MpuAQ1 (Figs 1r,s). Horn-like hyphae were described in static culture of M. purpurescens. No
arthroconidia were seen during observation.
   Thus, the studied cultures by micromorphological characters on agar and liquid media were not
differed significantly (Tables 2 and 3).

Growth rate and biomass formation
Mycelia of morels are the fastest growing among all taxonomic groups of mushrooms. In studied
Morchella collections the range of average growth rate (GRavr) was from 11.55 mm d-1 to 18.41
mm d-1 (Table 4). Mycelia, growing from the centre of the Petri dish, almost covers the entire plate
within 2-3 days. The highest GRavr was detected in M. importuna (18.41 and 16.25 mm d-1), then M.
esculenta (13.91-14.08 mm d-1), M. pururanscens MpuAQ1 (13.80 mm d-1), M. dunalii MduNU2-4
(13.40 mm d-1) and M. disparilis (13.30 mm d-1) cultures. Two strains of M. dunalii MduGR3–4 and
M. pururanscens MpuAQ1, showed relatively lower average growth rate values (11.70 mm d-1 and
11.55 mm d-1 ), respectively (Table 4).




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Table 4 - Mycelial average growth rate (GRavr, mm d-1) and biomass formation (g, g L-1) of Morchella collections on PDA
at 23 oC
 Species              Strain           GRavr ± SD        Biomass (g) 10th day ± SD        Biomass (g) 21st day ± SD
                                                         g                gL  -1
                                                                                          g                g L-1
 M. disparilis        MdiSU1-1         13.30 ± 0.42      0.08 ± 0.02      0.8 ± 0.02      0.76 ± 0.02      7.6 ± 0.24
 M. dunalii           MduNU2-4         13.40 ± 0.42      0.11 ± 0.01      1.1 ± 0.01      0.78 ± 0.02      7.8 ± 0.17
                      MduGR3-4         11.70 ± 0.32      0.07 ± 0.01      0.7 ± 0.01      0.47 ± 0.01      4.7 ± 0.05
 M. esculenta         MesAQ2-c         14.08 ± 0.83      0.06 ± 0.03      0.6 ± 0.03      0.74 ± 0.02      7.4 ± 0.17
                      MesAQ6-2         13.91 ± 0.97      0.06 ± 0.01      0.6 ± 0.01      0.74 ± 0.03      7.4 ± 0.26
                      MesFI2-3         13.91 ± 0.53      0.06 ± 0.01      0.6 ± 0.01      0.77 ± 0.03      7.7 ± 0.28
 M. importuna         MimBL1-1         18.41 ± 0.44      0.15 ± 0.02      1.5 ± 0.02      0.92 ± 0.03      9.2 ± 0.25
                      MimMO1-1         16.25 ± 0.80      0.14 ± 0.01      1.4 ± 0.01      0.86 ± 0.04      8.6 ± 0.43
 M. purpurascens      MpuAQ1           11.55 ± 0.11      0.09 ± 0.01      0.9 ± 0.01      0.45 ± 0.03      4.5 ± 0.34
                      MpuTN2           13.80 ± 0.35      0.14 ± 0.01      1.4 ± 0.01      0.67 ± 0.03      6.7 ± 0.28

    In our experimental conditions, Morchella collections formed around 0.9-1.5 g L-1 of dry biomass
in average during 10 days of surface cultivation (Table 4, Supplementary Fig. S4). During 21 days
of growth the cultures formed from 4.5 to 9.2 g L-1 of dry biomass. More than 6.0 g L-1 biomass was
accumulated all strains, except M. purpurascens MpuAQ1 (4.5 g L-1), which did not form sclerotia,
and M. dunalii MduGR3-4 (4.7 g L-1), possibly due to well-developed aerial mycelia. The strains
which developed substrate mycelia were accumulated from 6.7 to 8.6 g L-1 dry biomass. The highest
amount of biomass 1.5 and 9.2 g L-1 on 10th and 21th days of growth, respectively was accumulated by
M. importuna MimBL1-1 strain (Table 4, Supplementary Fig. S4).
    Thus, a similar growth trend between biomass and GRavr was found (Supplementary Fig. S4).
Mycelial GRavr, formation of biomass and development of sclerotia may indicate the aging cultures.
It is essential for commercial evaluation of mycelial strains to avoid usage of aging spawn in morel
cultivation.

Conclusion
Screening of cultural characteristics of five edible medicinal Morchella collections (M. esculenta,
M. dunalii, M. importuna, M. disparilis, M. purpurascens), such as colony morphology and
pigmentation, GRavr, development of vegetative (sclerotia) and asexual (arthroconidia) structures in
the life cycle, as well as biomass formation in liquid medium revealed their taxonomic significance.
Mycelial characteristics are required for proper taxonomic identification and commercial evaluation
of Morchella cultures during their biotechnological cultivation.

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