molecules
Review
The Psychrotolerant Antarctic Fungus
Lecanicillium muscarium CCFEE 5003: A Powerful
Producer of Cold-Tolerant Chitinolytic Enzymes
Massimiliano Fenice
Dipartimento di Scienze Ecologiche e Biologiche, University of Tuscia, Largo Università snc,
I-01100 Viterbo, Italy; [email protected]; Tel.: +39-0761-357-318
Academic Editor: Derek J. McPhee
Received: 26 February 2016; Accepted: 31 March 2016; Published: 5 April 2016
Abstract: Lecanicillium muscarium CCFEE 5003, isolated in Continental Antarctica, is a powerful
producer of extracellular cold-tolerant enzymes. Chitin-hydrolyzing enzymes seems to be the
principal extracellular catalytic activities of this psychrotolerant fungus. The production of chitinolytic
activities is induced by chitin and other polysaccharides and is submitted to catabolite repression.
The chitinolytic system of L. muscarium consists of a number of different proteins having various
molecular weights and diverse biochemical characteristics, but their most significant trait is the
marked cold-tolerance. L. muscarium and selected strains of the biocontrol agent of pathogenic
fungi Trichoderma harzianum, have been compared for their ability to produce chitinolytic enzymes at
different temperatures. At low temperatures the Antarctic strain was definitely much more efficient.
Moreover, the fungus was able to exert a strong mycoparasitic action against various other fungi
and oomycetes at low temperatures. The parasitic role of this organism appeared related to the
production of cell wall degrading enzymes being the release of extracellular chitinolytic enzymes
a key event in the mycoparasitic process. Due to the mentioned characteristics, L. muscarium could
have an important role for potential applications such as the degradation of chitin-rich materials at
low temperature and the biocontrol of pathogenic organisms in cold environments. For these reasons
and in view of future industrial application, the production of chitinolytic enzymes by the Antarctic
fungus has been up-scaled and optimised in bench-top bioreactor.
Keywords: Continental Antarctica; chitinolytic enzymes; Lecanicillium muscarium; cold-tolerant
enzymes
1. Introduction
Antarctica is generally considered the most extreme continent concerning temperature, winds,
altitude, isolation, UV-irradiation and dryness [1]. The very unhospitable Antarctic environment
requires high levels of adaptation and specialization. Adaptation is the fundamental topic in Antarctic
biology and the study of the biodiversity of Antarctic organisms is of paramount scientific importance:
Antarctica is a unique and precious source of genes that are only partially investigated [2].
Microorganisms dominate the Antarctic biology [3]. What adaptation strategies do Antarctic
microorganisms adopt? They must cope with combinations of highly stressful environmental
conditions and in particular low temperatures, wide temperature fluctuations, scarce water availability,
frequent freeze-thaw cycles and, in summer, intense solar radiation [4]. Most of them show
physiological parameters optima well above the average environmental temperature [4,5], even
if they still function close to freezing conditions where they are subjected to great increase of external
solutes concentration needing resistance to osmotic stress for survival [6]. Other adaptation strategies
include pigmentation to face high levels of radiation and peculiar fatty acid compositions to increase
membrane fluidity [7,8].
Molecules 2016, 21, 447; doi:10.3390/molecules21040447
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include pigmentation to face high levels of radiation and peculiar fatty acid compositions to increase
membrane fluidity [7,8].
The
The majority
majority of
of Antarctic
Antarctic microfungi
microfungi are
are psychrotolerant
psychrotolerantinstead
instead of
of psychrophilic
psychrophilic [5,9],
[5,9], showing
showing
better
adaptation
to
the
unstable
environment.
Here,
microorganisms
must
grow
whenever
water
better adaptation to the unstable environment. Here, microorganisms must grow whenever water
is
is
available
and
the
production
of
enzymes
having
broad
temperature
range
of
activity
would
be
available and the production of enzymes having broad temperature range of activity would be aa
winning
winningsurvival
survivalstrategy.
strategy.
Chitinolytic
Chitinolytic enzymes
enzymes have
have been
been widely
widely studied
studied and
and some
some of
of them,
them, produced
produced by
by fungi,
fungi, appear
appear
of
great
interest
for
industrial
or
environmental
applications.
Nevertheless,
there
is
still
necessity
of great interest for industrial or environmental applications. Nevertheless, there is still necessity of
of
cheap
cheap and
and commercially
commerciallysuitable
suitablesources
sourcesof
ofthese
theseenzymes.
enzymes.
The
hydrolysis of
ofchitin
chitinisisperformed
performed
a series
of chitinolytic
enzymes
thatgenerally
are generally
The hydrolysis
byby
a series
of chitinolytic
enzymes
that are
called
called
chitinases
using
a
misleading
term.
Their
current
classification,
supplied
by
the
Enzyme
chitinases using a misleading term. Their current classification, supplied by the Enzyme Commission
Commission
(EC), is inadequate
describe
theand
various
and diverse
activities
in Nature,
having
(EC), is inadequate
to describetothe
various
diverse
activities
found found
in Nature,
having
very
very
different
hydrolytic
action
on
the
polysaccharide
[10].
Only
two
classes
of
enzymes
are
listed,
different hydrolytic action on the polysaccharide [10]. Only two classes of enzymes are listed, being
being
the different
activities
now merged
in theEC
same
EC
class: chitinase,
(EC producing
3.2.1.14) producing
the different
activities
now merged
in the same
class:
chitinase,
(EC 3.2.1.14)
“random
“random
hydrolysis
of
N-acetyl-βD
-glucosaminide
(1
Ñ
4)-β-linkages
in
chitin
and chitodextrins”;
hydrolysis of N-acetyl-β-D-glucosaminide (1 → 4)-β-linkages in chitin and chitodextrins”;
and β-Nand
β-N-acetylhexosaminidase
(EC 3.2.1.52)
that “releases
N-acetylD -hexosamine
residues,
the
acetylhexosaminidase
(EC 3.2.1.52)
that “releases
N-acetylD-hexosamine
residues,
at theatnonnon-reducing
terminal,
from
chitin
and
chitodextrins”.
However,
numerous
authors
still
use
the
reducing terminal, from chitin and chitodextrins”. However, numerous authors still use the old
old
classification
making
actual
situationrather
ratherconfusing
confusingand
andambiguous
ambiguous [11,12].
[11,12]. The
classification
making
thethe
actual
situation
The enzymes
enzymes
acting
randomly
inside
the
polysaccharide,
generating
shorter
fragments,
are
frequently
defined
acting randomly inside the polysaccharide, generating shorter fragments, are frequently defined as
as
“endo-chitinases”,
“endo-chitinases”, while
while those
those acting
acting externally
externally are
are defined
defined as
as “exo-chitinases”
“exo-chitinases” [13–15].
[13–15]. Moreover,
Moreover,
other
other much
much specific
specific hydrolytic
hydrolytic mechanisms
mechanisms had
had been
been described
described (Figure
(Figure 1).
1). For
For example,
example, enzymes
enzymes
acting
only
on
chitobiose
are
called
“chitobiases”,
while
those
releasing
this
disaccharide
acting only on chitobiose are called “chitobiases”, while those releasing this disaccharide from
from chitin
chitin
or
or chitodextrins
chitodextrins are
arenamed
named“chitobiosidases”
“chitobiosidases”[13,16].
[13,16].
Figure 1.
1. Schematic
Schematic representation
representationof
of the
the catalytic
catalytic activity
activity by
by some
some chitinolytic
chitinolytic enzymes
enzymeson
on chitin
chitin and
and
Figure
chitobiose.
Only
chitinase
(EC
3.2.1.14)
and
β-N-acetylhexosaminidase
(EC
3.2.1.52)
are
described
by
chitobiose. Only chitinase (EC 3.2.1.14) and β-N-acetylhexosaminidase (EC 3.2.1.52) are described by
the Enzyme
Enzyme Commission.
Commission.
the
Conventionally,most
mostofofthese
thesehydrolases
hydrolasesfind
find
uses
chitin
hydrolysis
(including
treatment
Conventionally,
uses
inin
chitin
hydrolysis
(including
thethe
treatment
of
of chitin-rich
wastes),
production
of derivatives,
chitin derivatives,
protoplast
and of
biocontrol
of
chitin-rich
wastes),
production
of chitin
protoplast
formationformation
and biocontrol
pathogenic
pathogenic[17–21].
organisms
[17–21].some
Moreover,
some
innovativeinapplications
the industries
food and have
wine
organisms
Moreover,
innovative
applications
the food andinwine
industries
have been
successfully
tested at
the[21,22].
laboratory
level [21,22].
Nevertheless,
most of the
been
successfully
tested
at the laboratory
level
Nevertheless,
most
of the abovementioned
abovementioned
processes,
particularenvironmental
those concerning
environmental
problems
(i.e.,
on-field
processes,
in particular
those in
concerning
problems
(i.e., on-field
control of
pathogens
control
of pathogens
or degradation
chitin-rich
wastes),
are strongly
restricted
by adverse
or
degradation
of chitin-rich
wastes), areof
strongly
restricted
by adverse
environmental
conditions,
in
environmental
conditions, inaffecting
particular
low
affecting both
chitinolytic
particular
low temperature,
both
thetemperature,
chitinolytic organisms
and the
their
hydrolyticorganisms
enzymes.
and is
their
hydrolyticproblem
enzymes.
is a notorious
problem
for recognised
chitinolytic
This
a notorious
forThis
recognised
powerful
chitinolytic
organismspowerful
such as Trichoderma
˝
organisms[19,23]
such aseven
Trichoderma
harzianum
[19,23]were
evenshown
if, recently,
shown
be
harzianum
if, recently,
some strains
to be some
activestrains
at 10 were
C [24].
Thus,tothe
active at 10 of
°C a[24].
Thus, the availability
a psychrophilic or,
better, aable
psychrotolerant
organism,
availability
psychrophilic
or, better, of
a psychrotolerant
organism,
to release cold-active
able to release
cold-active
enzymes
very
low temperature
°C orother
below),
is important
chitinolytic
enzymes
at verychitinolytic
low temperature
(5 ˝ at
C or
below),
is important (5
where
microorganisms
where
microorganisms
fail.applications
This is particular
in many applications
the
fail.
Thisother
is particular
true in many
includingtrue
the hydrolysis
of chitin-rich including
wastes at low
hydrolysis of
chitin-rich
wastes
at low temperature
and the biocontrol
of pathogens
cold
temperature
and
the biocontrol
of pathogens
in cold environments.
In this context,
the search in
of new
chitinolytic organisms and/or enzymes is still of great scientific and applied interest [25].
Molecules 2016, 21, 447
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Among chitinolytic fungi, the genus Lecanicillium has been mainly studied for its involvement
in the pathogenesis of a wide array of invertebrates; “Lecanicilli” have a wide host range and have
been isolated from a variety of insects [26]. Lecanicillium muscarium, a good representative of this
genus, has also been isolated from insects (i.e., aphids, scales, whiteflies, thrips, planthoppers, etc.)
all over the world and its pathogenic activity against them proved [27–34]. Preparations containing
the fungus, active against whiteflies, thrips, aphids and mites, are commercialised worldwide as the
bio-pesticides “Mycotal” and “Verticillin” [35]. L. muscarium has also been isolated from arachnids
and nematodes [31,36]. The fungus exerts also its parasitic activity against various fungi involved in
plant diseases and its use as biocontrol agent has been demonstrated [31,37–40]. The mycoparasitism
against oomycetes has been demonstrated “in vitro” as well [19].
In this study, we will review the fascinating features of an emerging strain (CCFEE 5003) of
Lecanicillium muscarium isolated in Continental Antarctica and showing very high production of a wide
array of extracellular cold-active chitinolytic enzymes.
2. Lecanicillium muscarium CCFEE 5003
Lecanicillium muscarium (Petch) strain CCFEE 5003 Zare & W. Gams was previously affiliated to
Acremonium strictum W. Gams [5] then to Verticillium lecanii Zimm strain A3, [17]. The new genus
Lecanicillium W. Gams & Zare, was proposed to accommodate the majority of entomogenous or
fungicolous Verticillium (section Prostrata) species [41]. A detailed account of the species and the new
combinations are supplied by Zare and Gams [26].
Lecanicillium muscarium CCFEE 5003, isolated from moss collected in Victoria Land on the west
coast of the Ross Sea (Antarctica) [5], is stored in the Culture Collection of Fungi from Extreme
Environments (CCFEE:) of the Dipartimento di Scienze Ecologiche e Biologiche, University of Tuscia,
Viterbo, Italy.
The thermal preferences of the fungus showed its clear psychrotolerant behaviour, being its
optimum of growth at 25 ˝ C with no big differences within 20–28 ˝ C. The fungus was also able to
grow, albeit much slower, at 0 ˝ C. It is worth noting that L. muscarium sporulation occurred in the
whole temperature range from 0 to 28 ˝ C, showing its great ability to diffuse in the environment [5].
During a preliminary study, screening the production of extracellular enzymes by Antarctic fungi,
strain CCFEE 5003 was noted for its production of a number of hydrolases (amylases, cellulases,
proteases and lipases) but was selected for its high chitinolytic activity (ca. 300 IU/L, International
Units) in submerged shaken cultures [17,42].
3. Production and Characterization of Chitinolytic Enzymes by L. muscarium CCFEE 5003
The production of chitinolytic enzymes by L. muscarium, in shaken cultures, appeared to be
inducible by various forms of raw and purified chitin [17,19,43]. Even if to a lesser extent, induction
was also obtained with glucans but, on the other hand, glucanases were also produced when the
fungus grew on chitins, being the production of these two cell wall degrading enzymes (CWDE)
always correlated [19]. The production of chitinolytic enzymes appeared to be subjected to catabolite
repression: no activity was detected in media containing both glucose and chitin until complete glucose
depletion occurred. In addition, no activity was found in media containing glucose as the sole carbon
source. The chitinolytic enzymes of L. muscarium were mainly present inside the fungal cells during the
first 24 h of growth to be massively secreted thereafter [17]. Strain CCFEE 5003 was also able to grow
and release high levels of these enzymes in media containing crab or shrimp wastes. This characteristic
is particularly interesting in view of applications at the environmental/industrial level [10].
The enzymatic system of L. muscarium, related to chitin hydrolysis, is very complex and has
been studied in detail. In shaken flasks, the fungus was able to release at least five different enzymes
as revealed by electrophoresis polyacrylamide gel (PAGE) carried out under partially denaturing
conditions, followed by renaturation and stained for activity using glycol chitin as substrate. These
proteins showed molecular weights ranging from 20 to 45 kDa (Figure 2) and isoelectric points in the
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proteins showed molecular weights ranging from 20 to 45 kDa (Figure 2) and isoelectric points in the
range 4.5–5.5 pH [19]. Moreover, a chitobiose-hydrolysing protein, not revealed by the activity-gel,
range 4.5–5.5 pH [19]. Moreover, a chitobiose-hydrolysing protein, not revealed by the activity-gel,
was released [10]. The principal chitinolytic enzymes produced by L. muscarium were purified and
was released [10]. The principal chitinolytic enzymes produced by L. muscarium were purified and
biochemically characterised; all of them were also very active at low temperature [10,43]. A complex
biochemically characterised; all of them were also very active at low temperature [10,43]. A complex
enzymatic system, consisting of many different proteins, having complementary action on chitin, is
enzymatic system, consisting of many different proteins, having complementary action on chitin, is typical
typical of very aggressive fungi using their enzymes to attack other organisms. Others, using
of very aggressive fungi using their enzymes to attack other organisms. Others, using environmental
environmental chitin merely as a nutrient, could have a much simplified system with just one or few
chitin merely as a nutrient, could have a much simplified system with just one or few proteins.
proteins.
Figure 2. One-dimension activity PAGE for chitinolytic enzymes by Lecanicillium muscarium CCFEE
Figure 2. One-dimension activity PAGE for chitinolytic enzymes by Lecanicillium muscarium CCFEE
5003 produced in shaken cultures at different times: 24 h, intracellular activity; 48 and 72 h
5003 produced in shaken cultures at different times: 24 h, intracellular activity; 48 and 72 h extracellular
extracellular activity. Molecular weight markers are shown in the range 15–75 kDa (unpublished
activity. Molecular weight markers are shown in the range 15–75 kDa (unpublished picture related
picture related to [19]).
to [19]).
The first enzyme studied in the early works (CHI) [43] was purified by preparative isoelectric
The first enzyme studied in the early works (CHI) [43] was purified by preparative isoelectric
focusing and ion-exchange chromatography on Q-Sepharose. It was able to hydrolyse colloidal and
focusing and ion-exchange chromatography on Q-Sepharose. It was able to hydrolyse colloidal and
glycol chitin better than other forms of chitin; xylan and chitosan were scarcely degraded. Moreover,
glycol chitin better than other forms of chitin; xylan and chitosan were scarcely degraded. Moreover,
no activity was detected on cellulose and chitobiose. This protein (molecular weight of 45 kDa, in
no activity was detected on cellulose and chitobiose. This protein (molecular weight of 45 kDa, in
SDS-PAGE) appeared to be glycosylated and had an isoelectric point of 4.9. The optimal pH for its
SDS-PAGE) appeared to be glycosylated and had an isoelectric point of 4.9. The optimal pH for its
activity was 4.0; at pH 3.5 and 4.5 the enzyme retained 53% and 66% of the activity shown at the
activity was 4.0; at pH 3.5 and 4.5 the enzyme retained 53% and 66% of the activity shown at the
optimum, respectively. Enzyme stability was highest at pH 5.0. The purified enzyme was active from
optimum, respectively. Enzyme stability was highest at pH 5.0. The purified enzyme was active from
5 to 60 °C with maximum at 40 °C. It is worth noting that at 5 ˝°C, 50% of its maximum activity was
5 to 60 ˝ C with maximum at 40 ˝ C. It is worth noting
that at 5 C, 50% of its maximum activity was
2+, Mn2+, Mg2+ and EDTA (100 mM) was recorded.
still retained. In addition, strong inhibition by Zn2+
still retained. In addition, strong inhibition by Zn , Mn2+ , Mg2+ and EDTA (100 mM) was recorded.
Two other chitinolytic enzymes, CHI1 and CHI2, were purified and biochemically characterized
Two other chitinolytic enzymes, CHI1 and CHI2, were purified and biochemically
[10]. CHI1, having molecular weight of 61 kDa, showed optima at pH 5.5 and 45 °C; while CHI2,
characterized [10]. CHI1, having molecular weight of 61 kDa, showed optima at pH 5.5 and 45 ˝ C;
having molecular weight of 25 kDa, showed optima at pH 4.5 and 40 °C. Both enzymes
maintained
while CHI2, having molecular weight of 25 kDa, showed optima
at pH 4.5 and 40 ˝ C. Both enzymes
2+, Hg2+ and Cu2+. In addition, CHI2 was
high levels of activity at 5 °C and were
inhibited
by
Fe
maintained high levels of activity at 5 ˝ C and were inhibited by Fe2+ , Hg2+ and Cu2+ . In addition, CHI2
markedly sensitive to allosamidin. Both proteins could be classified as N-acetylhexosaminidases (E.C.
was markedly sensitive to allosamidin. Both proteins could be classified as N-acetylhexosaminidases
3.2.1.52), but showed different roles in chitin hydrolysis: CHI1 could be defined as a “chitobiase”
(E.C. 3.2.1.52), but showed different roles in chitin hydrolysis: CHI1 could be defined as a “chitobiase”
while CHI2 revealed a main “exo-chitinase” activity [10].
while CHI2 revealed a main “exo-chitinase” activity [10].
The preliminary molecular characterization of a gene coding for a chitinase (probably CHI) from
The preliminary molecular characterization of a gene coding for a chitinase (probably CHI)
L. muscarium CCFEE 5003 has been obtained sequencing a 736 bp DNA fragment [44]. This
from L. muscarium CCFEE 5003 has been obtained sequencing a 736 bp DNA fragment [44]. This
corresponded to 245 amino acids and represented ca. 60% of the whole protein sequence for the
corresponded to 245 amino acids and represented ca. 60% of the whole protein sequence for the
Lecanicillium spp. endochitinase (GH18, glycosyl hydrolases, family 18, 423 aa). However, according
Lecanicillium spp. endochitinase (GH18, glycosyl hydrolases, family 18, 423 aa). However, according to
to the comparison carried out in the NCBI GenBank database with BlastX, no match with Lecanicillium
the comparison carried out in the NCBI GenBank database with BlastX, no match with Lecanicillium
chitinolytic enzymes was obtained. By contrast, matching was achieved with various other fungal
chitinolytic enzymes was obtained. By contrast, matching was achieved with various other fungal
chitinases (Table 1). It is worth noting that, in all cases, although the query coverage was quite high
chitinases (Table 1). It is worth noting that, in all cases, although the query coverage was quite high
(93%–95%), the identities were always low (never exceeding 50%). These marked differences could
(93%–95%), the identities were always low (never exceeding 50%). These marked differences could be
be explained as an adaptation strategy adopted by the Antarctic fungus to produce enzymes active
explained as an adaptation strategy adopted by the Antarctic fungus to produce enzymes active in a
in a broad temperature range.
broad temperature range.
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Table 1. Comparison of the partial chitinase sequence of L. muscarium CCFEE 5003 with other fungal
chitinase
as obtained by
NCBI
GenBank
database
query
BlastX. Only
some
ofwith
the best
matches
Table 1. Comparison
of the
partial
chitinase
sequence
of with
L. muscarium
CCFEE
5003
other
fungal
are
shown.
chitinase as obtained by NCBI GenBank database query with BlastX. Only some of the best matches
are shown.
Description
Query Coverage Identity
Chitinase (Metarhizium album)
95%
42%
Description
Query Coverage
Identity
Chitinase (Hirsutella thompsonii)
94%
44%
Chitinase (Metarhizium album)
95%
42%
Chitinase (H. thompsonii)
94%
44%
Chitinase (Hirsutella thompsonii)
94%
44%
Endochitinase
33
(Tolypocladium
ophioglossoide)
94%
43%
Chitinase (H. thompsonii)
94%
44%
Chitinase
33 (Trichoderma
virens)
94%
41%
Endochitinase
33 (Tolypocladium
ophioglossoide)
94%
43%
Chitinase
33
(Trichoderma
virens)
94%
41%
Glycoside hydrolase family 18 (T. reesei)
94%
39%
Glycoside hydrolase
hydrolase family
1818
(T.(T.
reesei)
94%
39%
Glycoside
family
atroviride)
94%
42%
Glycoside hydrolase family 18 (T. atroviride)
94%
42%
Endochitinase
(T.atroviride)
atroviride)
94%
42%
Endochitinase (T.
94%
42%
Glycoside
family
virens)
94%
41%
Glycoside hydrolase
hydrolase family
1818
(T.(T.
virens)
94%
41%
Chitinase (T.
(T. virens)
94%
41%
Chitinase
virens)
94%
41%
Chitinase
3
(Escovopsis
weberi)
94%
36%
Chitinase 3 (Escovopsis weberi)
94%
36%
Putative Chitinase (Torrubiella hemipterigena)
93%
50%
Putative Chitinase (Torrubiella hemipterigena)
93%
50%
Related to endochitinase (Claviceps purpurea)
93%
49%
Related to endochitinase (Claviceps purpurea)
93%
49%
Accession N°
KHN99830.1 ˝
Accession N
AIT18903.1
KHN99830.1
AIT18889.1
AIT18903.1
KND91481.1
AIT18889.1
ACJ04784.1
KND91481.1
ACJ04784.1
XP_006961069.1
XP_006961069.1
XP_013947241.1
XP_013947241.1
ABO38127.1
ABO38127.1
XP_013958614.1
XP_013958614.1
ABP96986.1
ABP96986.1
KOS22932.1
KOS22932.1
CEJ90418.1
CEJ90418.1
CCE32650.1
CCE32650.1
4. Potential Applications of the Chitinolytic Enzymes by L. muscarium CCFEE 5003
4. Potential Applications of the Chitinolytic Enzymes by L. muscarium CCFEE 5003
Since cell walls of most pathogenic or spoiling microorganisms contain chitin, the degradation of
Since cell walls of most pathogenic or spoiling microorganisms contain chitin, the degradation
which affects their growth and differentiation, chitinolytic enzymes could play a fundamental role in
of which affects their growth and differentiation, chitinolytic enzymes could play a fundamental role
the control of these organisms [43]. The above mentioned features suggested possible applications of
in the control of these organisms [43]. The above mentioned features suggested possible applications
the Antarctic strain and its enzymes.
of the Antarctic strain and its enzymes.
The purified CHI showed evident inhibitory effects on a series of moulds involved in refrigerated
The purified CHI showed evident inhibitory effects on a series of moulds involved in
food spoilage (Mucor plumbeus, Cladosporium cladosporoides, Penicillium verrucosum and Aspergillus
refrigerated food spoilage (Mucor plumbeus, Cladosporium cladosporoides, Penicillium verrucosum and
versicolor). The enzyme caused mycelial damages, cell lysis, inhibition of conidia germination,
Aspergillus versicolor). The enzyme caused mycelial damages, cell lysis, inhibition of conidia
formation and bursting of protoplast (Figure 3). The results of this study would suggest that the
germination, formation and bursting of protoplast (Figure 3). The results of this study would suggest
chitinolytic enzyme could be employed in place or in combination with traditional food preservatives,
that the chitinolytic enzyme could be employed in place or in combination with traditional food
the use of which has been widely criticized [43], as already proposed in food technology for other
preservatives, the use of which has been widely criticized [43], as already proposed in food
enzymatic preparations containing chitinolytic enzymes [22].
technology for other enzymatic preparations containing chitinolytic enzymes [22].
Figure 3. Cultures of Mucor plumbeus treated with the purified chitinolytic enzyme (CHI) of L.
Figure 3. Cultures of Mucor plumbeus treated with the purified chitinolytic enzyme (CHI) of L. muscarium
muscarium CCFEE 5003 and showing protoplast formation, with traces of degraded cell wall, (A) and
CCFEE 5003 and showing protoplast formation, with traces of degraded cell wall, (A) and bursting (B)
bursting (B) (unpublished picture related to [43]).
(unpublished picture related to [43]).
The use of purified enzymes in large scale applications is expensive and sometime not necessary,
The use of purified enzymes in large scale applications is expensive and sometime not necessary,
while crude or semi-purified enzyme preparations could represent a feasible solution. The crude
while crude or semi-purified enzyme preparations could represent a feasible solution. The crude
CWDE produced by L. muscarium have been applied on red and white grape bunches inoculated with
CWDE produced by L. muscarium have been applied on red and white grape bunches inoculated with
spores of Aspergillus carbonarius, a well-known producer of ochratoxin A, in order to verify possible
spores of Aspergillus carbonarius, a well-known producer of ochratoxin A, in order to verify possible
reduction of the contamination by the toxigenic fungus in post-harvest conditions. Presence of the
Molecules 2016, 21, 447
Molecules 2016, 21, 477
6 of 13
6 of 13
contaminant
control (untreated
wasfungus
very high
(>2000 CFU/mL);
by contrast,
on treated
reduction of in
thethe
contamination
by thegrapes)
toxigenic
in post-harvest
conditions.
Presence
of the
fruits,
it was reduced
by 89(untreated
and 95%, for
red and
white
respectively.
Light
contaminant
in the control
grapes)
was
verygrape,
high (>2000
CFU/mL);
bymicroscopy
contrast, onshowed
treated
that
the
solution
A. red
carbonarius
spores
therespectively.
same damages
in Figure
3
fruits,
it enzymes
was reduced
by 89induced
and 95%,infor
and white
grape,
Lightdescribed
microscopy
showed
[21].
that the enzymes solution induced in A. carbonarius spores the same damages described in Figure 3 [21].
The high cold tolerance of L. muscarium and its enzymes could be exploited in view of possible
applications
applications as
as a biocontrol organism for phytopathogens in cold environments. Thus,
Thus, the fungus
was compared with two selected strains of Trichoderma harzianum (strains P1 and T22), a well-known
chitinolytic
chitinolytic fungus
fungus commercially
commercially used
used for
for the
the above
above applications
applications but
but showing
showing limitations
limitations at
at low
low
temperatures
temperatures [23,45]. The
The three
three strains
strains were
were grown
grown at
at 5, 15 and 25 ˝°C
C under inducing conditions
(Figure 4).
Figure
Figure 4.
4. Chitinolytic
Chitinolytic activity
activity of
of L.
L. muscarium
muscarium CCFEE
CCFEE 5003
5003 (black
(black lines),
lines), T.
T. harzianum
harzianum P1
P1 (green
(green lines)
lines)
˝
and
T.
harzianum
T22
(red
lines)
at
5,
15
and
25
°C.
(Modified
from
[43]).
and T. harzianum T22 (red lines) at 5, 15 and 25 C. (Modified from [43]).
˝ C, for
At
At 25
25 °C,
for both
both L.
L. muscarium
muscarium and
and T.
T. harzianum
harzianum T22,
T22, the
the time
time course
course of
of the
the enzyme
enzyme activity
activity was
was
quite
reaching aa maximum
maximum (ca
(ca 230
230 IU/L)
IU/L) after
quite similar,
similar, reaching
after 72
72 hh of
of incubation.
incubation. Strain
Strain P1
P1 performance
performance was
was
˝ C,the
definitely
lower reaching
reaching only
only 121
121 IU/L
IU/L after
definitely lower
after 120
120 h.
h. At
At 15
15 °C,
theperformance
performance of
of the
the three
three strains
strains was
was
˝
˝
quite
similar
to
that
recorded
at
25
°C.
At
5
°C,
the
enzyme
activity
of
L.
muscarium
was
very
similar
quite similar to that recorded at 25 C. At 5 C, the enzyme activity of L. muscarium was very similar
to
˝ C°C
to
that
obtained
(203IU/L
IU/Lafter
after144
144h).
h). On
On the
the contrary,
contrary, the
the production
production of
that
obtained
at at
2525
(203
of the
the two
two Trichoderma
Trichoderma
strains
and 57
57 IU/L,
IU/L, for
strains was
was definitely
definitely lower
lower (22
(22 and
forstrain
strainT22
T22and
andP1,
P1, respectively)
respectively) [43].
[43].
5. Optimization
Optimizationof
ofthe
theChitinolytic
ChitinolyticEnzymes
EnzymesProduction
Productionin
inBioreactors
Bioreactors
In view
view of
ofpossible
possibleapplications
applicationsatat
industrial
level,
microorganisms
be cultivated
in
thethe
industrial
level,
microorganisms
mustmust
be cultivated
in bulk.
bulk.
possible
industrial
strainshow
must good
showcapability
good capability
grow
and produce
the
Thus, Thus,
every every
possible
industrial
strain must
to growtoand
produce
the studied
studied
metabolites
in bioreactors.
all microorganisms
this characteristic
enzymesenzymes
and/or and/or
metabolites
in bioreactors.
Not allNot
microorganisms
possesspossess
this characteristic
that is
that
is mandatory
for process
every process
scale-up.
mandatory
for every
scale-up.
In this context, the production of chitinolytic enzymes by strain CCFEE 5003, after preliminary
tests in shaken flasks, was optimised in bench-top
bench-top bioreactors
bioreactors using
using both
both purified
purified chitin
chitin (colloidal)
(colloidal)
and chitin-rich materials obtained from crab and shrimp shells wastes.
Response Surface
SurfaceMethodology
Methodology(RSM)
(RSM)
was
employed
to investigate
the effects
of combining
was
employed
to investigate
the effects
of combining
stirrer
stirrer
speed
(in the
rangerpm)
200–500
rpm) and
rate (in
the vvm)
range[46].
0.5–1.5
vvm) [46].
speed (in
the range
200–500
and aeration
rateaeration
(in the range
0.5–1.5
Optimization
of
Optimization
of these
fundamental
parameters
was
carried out
quantitative and
these fundamental
process
parametersprocess
was carried
out using
quantitative
andusing
quantitative-multilevel
quantitative-multilevel factors for aeration and agitation, respectively. The model (quadratic Doptimal) revealed high reliability and good statistical performance (R2, 0.931; Q2, 0.869). Maximum of
Molecules 2016, 21, 447
7 of 13
factors for aeration and agitation, respectively. The model (quadratic D-optimal) revealed high
7 of 13
reliability and good statistical performance (R2 , 0.931; Q2 , 0.869). Maximum of activity (373.0 IU/L),
under optimised
conditions,
was predicted
at an intermediate
stirring
(ca. 327 rpm)
and 1.1
activity
(373.0 IU/L),
under optimised
conditions,
was predicted
at anrate
intermediate
stirring
ratevvm.
(ca.
However,
the
subsequent
experimental
confirmation
of
the
model
indicated
that
highest
activity
327 rpm) and 1.1 vvm. However, the subsequent experimental confirmation of the model indicated
(383.7
IU/L)activity
was achieved
at 1 was
vvmachieved
and 300atrpm.
Clearly,
enzyme
production
was
strongly
that highest
(383.7 IU/L)
1 vvm
and 300the
rpm.
Clearly,
the enzyme
production
affected
by the
shear effects
by produced
high agitation
andagitation
aerationand
rates
(Figurerates
5). In(Figure
bioreactors,
was
strongly
affected
by the produced
shear effects
by high
aeration
5). In
under
optimised
conditions,
the
fungus
produced
a
number
of
chitinolytic
enzymes
higher
those
bioreactors, under optimised conditions, the fungus produced a number of chitinolyticthan
enzymes
released
in shaken
flasks; moreover,
was 23%
higher. was 23% higher.
higher
than
those released
in shakenproduction
flasks; moreover,
production
Molecules 2016, 21, 477
Figure 5. Combined effects of agitation and aeration on the chitinolytic activity of L. muscarium CCFEE
Figure 5. Combined effects of agitation and aeration on the chitinolytic activity of L. muscarium CCFEE
5003 cultivated in bioreactor as predicted by RSM. Colour codes in the surface response indicate
5003 cultivated in bioreactor as predicted by RSM. Colour codes in the surface response indicate
different ranges of values (unpublished picture related to [46]).
different ranges of values (unpublished picture related to [46]).
This work, demonstrating the capacity of L. muscarium CCFEE 5003 to grow in bench-top
This work, demonstrating the capacity of L. muscarium CCFEE 5003 to grow in bench-top
bioreactors, identified the optimal conditions for the possible upscale of production at the industrial
bioreactors, identified the optimal conditions for the possible upscale of production at the industrial
level [46].
level [46].
On raw wastes from shrimp and crab manufacture, the production of chitinolytic enzymes by
On raw wastes from shrimp and crab manufacture, the production of chitinolytic enzymes by
the fungus (in shaken flasks) was much lower than that obtained using colloidal chitin. This was an
the fungus (in shaken flasks) was much lower than that obtained using colloidal chitin. This was an
expected result, since colloidal chitin is a purified substrate with stronger inducing effects. In
expected result, since colloidal chitin is a purified substrate with stronger inducing effects. In addition,
addition, the two wastes contained traces of other nutrients, such as fats and proteins, that could
the two wastes contained traces of other nutrients, such as fats and proteins, that could activate some
activate some repression mechanisms. However, since the production on shrimp wastes was much
repression mechanisms. However, since the production on shrimp wastes was much higher than that
higher than that on crab shells (104.6 and 48.6 IU/L, respectively) optimization in bioreactor was
on crab shells (104.6 and 48.6 IU/L, respectively) optimization in bioreactor was carried out using the
carried out using the first substrate only. In this case, optimization in bioreactor by RSM was carried
first substrate only. In this case, optimization in bioreactor by RSM was carried out to find best pH and
out to find best pH and substrate concentration under the optimised conditions of agitation and
substrate concentration under the optimised conditions of agitation and aeration previously reported.
aeration previously reported. The optimization process permitted to improve the production by 137%
The optimization process permitted to improve the production by 137% (243.6 IU/L).
(243.6 IU/L).
The ability of strain CCFEE 5003 to produce high level of chitinolytic enzymes in submerged
The ability of strain CCFEE 5003 to produce high level of chitinolytic enzymes in submerged
processes was not accompanied by same result when the fungus was grown in solid-state bioreactors
processes was not accompanied by same result when the fungus was grown in solid-state bioreactors
(unpublished results). This was somehow an unexpected result since Lecanicilli are known to be quite
(unpublished results). This was somehow an unexpected result since Lecanicilli are known to be quite
good producers of these enzymes also when employing this technology [18,47].
good producers of these enzymes also when employing this technology [18,47].
6. Mycoparasitic Action of L. muscarium
6. Mycoparasitic Action of L. muscarium
Bruce and co-workers [48] described mycoparasitism as an antagonistic interaction between two
Bruce and co-workers [48] described mycoparasitism as an antagonistic interaction between two
fungal organisms during which the parasite establishes intimate contacts with the host prior to release
fungal organisms during which the parasite establishes intimate contacts with the host prior to
CWDE. Other authors [49–51] correlated mycoparasitism to well-defined events, such as coiling around
release CWDE. Other authors [49–51] correlated mycoparasitism to well-defined events, such as
and penetration into the host, also mentioning the release of lytic enzymes. Nevertheless, most authors
coiling around and penetration into the host, also mentioning the release of lytic enzymes.
agree that stable contacts between the two organisms must happen [49–51]. Some mycoparasites (i.e.,
Nevertheless, most authors agree that stable contacts between the two organisms must happen [49–
51]. Some mycoparasites (i.e., Trichoderma spp.) produce extracellular inhibiting metabolites and/or
volatile compounds with no physical contact with the host [51].
Molecules 2016, 21, 447
8 of 13
Trichoderma spp.) produce extracellular inhibiting metabolites and/or volatile compounds with no
Molecules 2016, 21, 477
8 of 13
physical
contact with the host [51].
Molecules 2016, 21, 477
8 of 13
L.
L. muscarium
muscarium CCFEE
CCFEE 5003
5003 always
always established
established firm
firm contact
contact with
with the
the host
host mycelia
mycelia after
after an
an initial
initial
phase
during
which
inhibitory
compounds
were
probably
released.
In
all
cases,
the
contact
with
the
L.during
muscarium
CCFEE
5003 always
established
firm contact
with In
theallhost
mycelia
after an
initial
phase
which
inhibitory
compounds
were probably
released.
cases,
the contact
with
the
host
caused
its
disruption.
phase
during
which
inhibitory
compounds
were
probably
released.
In
all
cases,
the
contact
with
the
host caused its disruption.
˝ C)
of the
strain occurred
occurred in
in aa wide
host The
caused
its disruption.of
The mycoparasitism
mycoparasitism
the Antarctic
Antarctic strain
wide range
range of
of temperatures
temperatures (5–25
(5–25 °C)
and
was
tested
plate
co-cultures
with
moulds
(Aspergillus
versicolor,
The
mycoparasitism
ofthrough
the Antarctic
occurred
wide of
range
of temperatures
(5–25 °C)
and it
it
was
tested “in
“in vitro”
vitro”
through
plate strain
co-cultures
withinaaaseries
series
of
moulds
(Aspergillus versicolor,
Botrytis
cinerea,
Cladosporium
cladosporoides,
Penicillium
verrucosum,
Mucor
mucedo
and
M.
plumbeus)
and
it
was
tested
“in
vitro”
through
plate
co-cultures
with
a
series
of
moulds
(Aspergillus
versicolor,
Botrytis cinerea, Cladosporium cladosporoides, Penicillium verrucosum, Mucor mucedo and M. plumbeus)
and
(Pythium
Phytophthora
palmivora)
food
spoilage
or
Botrytis
cinerea, Cladosporium
cladosporoides, and
Penicillium
verrucosum,
Mucorcausing
mucedo and
plumbeus)
and oomycetes
oomycetes
(Pythium aphanidermatum
aphanidermatum
and
Phytophthora
palmivora)
causing
foodM.
spoilage
or
phytopathogenicity
[19,43].
and
oomycetes (Pythium
phytopathogenicity
[19,43].aphanidermatum and Phytophthora palmivora) causing food spoilage or
In
common
phytopathogenicity
[19,43].
In all
all co-cultures,
co-cultures,
common features
features were
were recorded.
recorded. The
The possible
possible hosts
hosts grew
grew well
well up
up to
to 3–10
3–10 mm
mm
from
L.
muscarium
colonies,
when
entered
an
“inhibition
zone”
(Figure
6).
At
this
phase,
all
co-cultures,
common
features
were
Thezone”
possible
hosts6).
grew
to 3–10
mm
fromInL.all
muscarium
colonies,
when
entered
anrecorded.
“inhibition
(Figure
At well
this up
phase,
all host
host
organisms
terminated
their
while
alterations
occurred
mycelia.
from
L. muscarium
colonies,
when entered
an “inhibition
zone”
(Figureto
6).their
At this
phase,Mycelial
all host
organisms
terminated
their growth
growth
while various
various
alterations
occurred
to
their
mycelia.
Mycelial
alterations:
(branching,
vacuolation,
cell-wall
damages
and
presence
of
abnormal
structures)
were
organisms
growth while
various
alterations
occurred
to their mycelia.
Mycelial
alterations: terminated
(branching,their
vacuolation,
cell-wall
damages
and presence
of abnormal
structures)
were
never
observed
in
axenic
cultures
(Figure
7).
Protoplast
formation
became
sometime
evident.
alterations:
(branching,
cell-wall
damages formation
and presence
of abnormal
never observed
in axenicvacuolation,
cultures (Figure
7). Protoplast
became
sometimestructures)
evident. were
never observed in axenic cultures (Figure 7). Protoplast formation became sometime evident.
Figure 6. Left: dual culture of L. muscarium (A) and Botrytis cinerea (B). Right: detail of a dual culture
Figure 6. Left: dual culture of L. muscarium (A) and Botrytis cinerea (B). Right: detail of a dual culture
Figure
6. Left: dual
culture
of plumbeus
L. muscarium
(A)contact
and Botrytis
cinerea (B).
detail(d)
of overgrowth
a dual culture
of L. muscarium
(a) and
Mucor
(b); (c)
zone between
theRight:
two fungi;
of
of L. muscarium (a) and Mucor plumbeus (b); (c) contact zone between the two fungi; (d) overgrowth
of
muscarium
Mucor(Right:
plumbeus
(b); (c) contact
zonerelated
between
the two
fungi;
(d) modified
overgrowth
of
L. L.
muscarium
on(a)
M.and
plumbeus
unpublished
picture
to [43].
Left:
picture
from
of L. muscarium on M. plumbeus (Right: unpublished picture related to [43]. Left: picture modified
L.
muscarium on M. plumbeus (Right: unpublished picture related to [43]. Left: picture modified from
[43]).
from [43]).
[43]).
Figure 7. Branching (left) and vacuolated (right) mycelia of Mucor mucedo. Grown in dual culture
Figure
Branching
(left) and
vacuolated
(right) mycelia of Mucor mucedo. Grown in dual culture
with L. 7.
muscarium
(modified
from
[19]).
Figure 7. Branching (left) and vacuolated (right) mycelia of Mucor mucedo. Grown in dual culture with
with L. muscarium (modified from [19]).
L. muscarium (modified from [19]).
All this suggested that L. muscarium released diffusible inhibiting compounds (lytic enzymes
Allantibiotics)
this suggested
thatmedium.
L. muscarium
diffusible
inhibiting compounds
enzymes
and/or
into the
This isreleased
typical of
other mycoparasitic
fungi such (lytic
as Trichoderma
Allantibiotics)
this suggested
thatmedium.
L. muscarium
released
diffusible
inhibiting compounds
(lytic
enzymes
and/or
into
the
This
is
typical
of
other
mycoparasitic
fungi
such
as
Trichoderma
spp. [48,50,51] or Verticillium spp. [40]. The low molecular weight chitinolytic enzyme, shown in
and/or
antibiotics)
into the spp.
medium.
Thislow
is molecular
typical of other chitinolytic
mycoparasitic
fungishown
such as
spp.
[48,50,51]
[40]. protein
The
enzyme,
in
Figure
2, couldor
beVerticillium
a possible diffusible
candidate; noweight
tests had been carried
out regarding
Figure
2,
could
be
a
possible
diffusible
protein
candidate;
no
tests
had
been
carried
out
regarding
possible production of antibiotics. It is worth nothing that the mycelial damages were also observed
possible
production
of antibiotics.
It is worth
nothing
that the
damages
also
observed
when crude
preparation
of L. muscarium
CWDE
was added
tomycelial
replace the
funguswere
in the
Petri
dishes
when
crudethe
preparation
of L. muscarium CWDE was added to replace the fungus in the Petri dishes
containing
host organisms.
containing the host organisms.
Molecules 2016, 21, 447
9 of 13
Trichoderma spp. [48,50,51] or Verticillium spp. [40]. The low molecular weight chitinolytic enzyme,
shown in Figure 2, could be a possible diffusible protein candidate; no tests had been carried out
regarding possible production of antibiotics. It is worth nothing that the mycelial damages were also
observed when crude preparation of L. muscarium CWDE was added to replace the fungus in the Petri
dishes containing the host organisms.
However,
Molecules
2016,after
21, 477some time, aerial mycelium of L. muscarium went in contact with the 9hosts
of 13 and,
shortly thereafter, overwhelmed them. Samples from the contact zones of the plate co-cultures were
after
some time, and
aerial
mycelium
of L. muscarium
in contact strain
with the
hosts
observedHowever,
under light
microscopy
SEM.
The contact
betweenwent
the Antarctic
and
theand,
host was
shortly
thereafter,
overwhelmed
them.
Samples
from
the
contact
zones
of
the
plate
co-cultures
always very firm. Coiling was observed, but coils were less regularly developed around hostwere
hyphae if
observed under light microscopy and SEM. The contact between the Antarctic strain and the host
compared
with other mycoparasites [49,50]. The typology of contact exerted by L. muscarium changed
was always very firm. Coiling was observed, but coils were less regularly developed around host
in relation to the host organism. With fungi, the it followed a typical sequence of events including
hyphae if compared with other mycoparasites [49,50]. The typology of contact exerted by L.
attachment
to the
host, in
producing
pressure,
release
and apenetration
into the
muscarium
changed
relation tomechanical
the host organism.
With
fungi,of
theCWDE
it followed
typical sequence
of host
mycelium
[38,43].
events including attachment to the host, producing mechanical pressure, release of CWDE and
With oomycetes,
no evident
penetration
into thedifferently,
host mycelium
[38,43]. penetration but firm adhesion and possibly mechanical
pressure occurred.
Since differently,
the activitynoofevident
glucanases
by L. muscarium
was lower
than that
of chitinolytic
With oomycetes,
penetration
but firm adhesion
and possibly
mechanical
pressure
occurred.
Since
the
activity
of
glucanases
by
L.
muscarium
was
lower
than
that
of
chitinolytic
enzymes [19], lack of penetration could probably due to the production of CWDE having reduced action
enzymes
[19], lack
penetration
could
probably
due to
the production
of CWDEmycelium
having reduced
on the
oomycetes
cell of
wall
with high
glucan
content.
Moreover,
L. muscarium
sometime
action
on
the
oomycetes
cell
wall
with
high
glucan
content.
Moreover,
L.
muscarium
mycelium
appeared almost fused with that of the host. In all cases, at the late stages of the interaction,
complete
sometime appeared almost fused with that of the host. In all cases, at the late stages of the interaction,
destruction of the host, that appeared strongly deflated and invaded by the parasite, was observed.
complete destruction of the host, that appeared strongly deflated and invaded by the parasite, was
Abundant sporulation of L. muscarium was recorded also in this case (Figure 8).
observed. Abundant sporulation of L. muscarium was recorded also in this case (Figure 8).
Figure 8. Top (A) Intermediate mycoparasitic stage of L. muscarium against Phytophtora palmivora:
Figure 8. Top (A) Intermediate mycoparasitic stage of L. muscarium against Phytophtora palmivora:
arrows show the contact between the thin mycelium of the Antarctic fungus and a P. palmivora
arrows
show thes contact
the thin (B)
mycelium
of the Antarctic
and a against
P. palmivora
sporangium;
= sporesbetween
of L. muscarium.
Late mycoparasitic
stage offungus
L. muscarium
sporangium;
s
=
spores
of
L.
muscarium.
(B)
Late
mycoparasitic
stage
of
L.
muscarium
against
P. palmivora; the oomycete appeared completely overwhelmed by the fungus. Bottom. (C)
P. palmivora;
the oomycete
appeared
overwhelmed
by mucedo:
the fungus.
Bottom. fungus
(C) Intermediate
Intermediate
mycoparasitic
stage completely
of L. muscarium
against Mucor
the Antarctic
(thin
mycoparasitic
of L. firm
muscarium
mucedo:
the Antarctic
fungus (thin
mycelium) stage
establishes
contact against
with theMucor
host (thick
mycelium)
and penetrates
it. (D)mycelium)
Late
mycoparasitic
stage of
L. muscarium
against
M. mucedo:and
the penetrates
host appears
overwhelmed. stage
establishes
firm contact
with
the host (thick
mycelium)
it.completely
(D) Late mycoparasitic
sporulation
of mucedo:
L. muscarium
is always
recorded
(modified
from [19]).
of L. Extensive
muscarium
against M.
the host
appears
completely
overwhelmed.
Extensive sporulation
of L. muscarium is always recorded (modified from [19]).
As for the action of the Antarctic fungus on yeasts, preliminary data (Fenice et al., unpublished
results) showed that, on co-cultures with Candida vinaria, the fungus behaviour was very similar to
that described above: after some time, it overgrew the yeast. However, microscopic observations
showed that the yeast was not completely destroyed by the fungus. Apparently, no contact was
recorded between the two organisms, but many yeast cells appeared abnormally bigger and showed
Molecules 2016, 21, 447
10 of 13
As for the action of the Antarctic fungus on yeasts, preliminary data (Fenice et al., unpublished
results) showed that, on co-cultures with Candida vinaria, the fungus behaviour was very similar to that
described above: after some time, it overgrew the yeast. However, microscopic observations showed
that the yeast was not completely destroyed by the fungus. Apparently, no contact was recorded
Molecules 2016, 21, 477
10 of 13
between the two organisms, but many yeast cells appeared abnormally bigger and showed damaged
cell walls. cell
In addition,
cells were
modified and
traces ofand
cell traces
lysis were
evident
damaged
walls. Inyeast
addition,
yeastmorphologically
cells were morphologically
modified
of cell
lysis
(Figure
9).
Likely,
the
fungus
released
some
CWDE
affecting
the
yeasts
but
their
concentration
and/or
were evident (Figure 9). Likely, the fungus released some CWDE affecting the yeasts but
their
composition was
not appropriate
to was
produce
disruption.
Further
experiments
are needed
to
concentration
and/or
composition
not complete
appropriate
to produce
complete
disruption.
Further
understand
possible
parasitic
role
of
L.
muscarium
against
yeasts
and
its
applied
potentials.
However,
experiments are needed to understand possible parasitic role of L. muscarium against yeasts and its
it is worth
noting that
the fungus
been
proved
exert
effective
mycoparasitism
against
black
applied
potentials.
However,
it is has
worth
noting
thattothe
fungus
has been
proved to exert
effective
yeasts
(black meristematic
fungi)
such(black
as Cryomyces
spp. [52].
mycoparasitism
against black
yeasts
meristematic
fungi) such as Cryomyces spp. [52].
Figure 9. Light microscopy of Candida vinaria from a dual culture with L. muscarium. Most yeast cells
Figure 9. Light microscopy of Candida vinaria from a dual culture with L. muscarium. Most yeast cells
appeared damaged and/or bigger than usual (Fenice et al., unpublished results).
appeared damaged and/or bigger than usual (Fenice et al., unpublished results).
7. Conclusions
7. Conclusions
This work summarises more than 20 years of investigations carried out on the chitinolytic fungus
This work summarises more than 20 years of investigations carried out on the chitinolytic
Lecanicillium muscarium CCFEE 5003 isolated from samples collected in Continental Antarctica in the
fungus Lecanicillium muscarium CCFEE 5003 isolated from samples collected in Continental Antarctica
early nineties. In particular, we describe its ability to release a number of cold-active extracellular
in the early nineties. In particular, we describe its ability to release a number of cold-active
chitinolytic enzymes and its strong action as mycoparasite against fungi and oomycetes. This
extracellular chitinolytic enzymes and its strong action as mycoparasite against fungi and oomycetes.
microorganism could be considered as one of the most promising “new” organisms isolated from
This microorganism could be considered as one of the most promising “new” organisms isolated
extreme environments and deserves to be further investigated. Actually, due to its very interesting
from extreme environments and deserves to be further investigated. Actually, due to its very
features, it would merit being exploited both at the industrial level, for the production of cold-tolerant
interesting features, it would merit being exploited both at the industrial level, for the production of
enzymes, reuse of chitin-rich wastes, and for environmental applications, in biocontrol of pathogens
cold-tolerant enzymes, reuse of chitin-rich wastes, and for environmental applications, in biocontrol
due to its strong mycoparasitic activity. However, prior to introducing the Antarctic organism in
of pathogens due to its strong mycoparasitic activity. However, prior to introducing the Antarctic
different ecosystems, tests must be done to verify possible negative environmental and/or ecological
organism in different ecosystems, tests must be done to verify possible negative environmental and/or
effects.
ecological effects.
Acknowledgments: The studies on Lecanicillium muscarium CCFEE 5003 mentioned in this short review were
Acknowledgments: The studies on Lecanicillium muscarium CCFEE 5003 mentioned in this short review were
partially supported
supported by
by the
the Italian
Italian PNRA
PNRA (Programma
(Programma Nazionale
Nazionale Ricerche
Ricerche Antartiche).
Antartiche).
partially
Conflicts
of Interest:
Interest: The
The author
author declares
declares that
that for
for this
this work
work there
there is
is no
no conflict
conflictof
ofinterest.
interest.
Conflicts of
References
References
1.
2.
3.
4.
5.
Vishniac, H.S.
H.S. The
Themicrobiology
microbiologyofofAntarctic
Antarctic
soils.
Antarctic
Microbiology;
Friedmann,
Wileysoils.
InIn
Antarctic
Microbiology;
Friedmann,
E.I.,E.I.,
Ed.;Ed.;
Wiley-Liss:
Liss:
NY, USA,
pp. 297–341.
New New
York,York,
NY, USA,
1993; 1993;
pp. 297–341.
Onofri, S.; Pagano,
Pagano S.; Zucconi,
Zucconi L.; Cicalini,
Cicalini AR.;
A.R.;Selbmann
Selbmann,L.;
L.;Fenice
Fenice,M.
M.Microfungi
Microfungiin
in Antarctic
Antarctic extreme
environments.
environments. Extracellular
Extracellular enzyme
enzyme activities.
activities. In
InProceedings
Proceedingsof
of the
the IBC’s
IBC’s World
World Congress on Enzyme
Technologies,
San Francisco,
Francisco, CA,
CA, USA,
USA, 10–12
10–12 March
March 1999.
1999.
Technologies, San
Friedmann, E.I.; Thistle, A.B. Foreword. In Antarctic Microbiology. Friedmann, E.I., Ed.; Wiley-Liss: New
York, NY, USA, 1993; pp. ix–x.
Vincent, W.F.; Howard-Williams, C.; Broady, P.A. Microbial communities and processes in Antarctic
flowing waters. In Antarctic Microbiology; Friedmann, E.I., Ed.; Wiley-Liss: New York, NY, USA, 1993; pp.
544–569.
Zucconi, L.; Pagano, S.; Fenice, M.; Selbmann, L.; Tosi, S.; Onofri, S. Growth temperature preferences of
Molecules 2016, 21, 447
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
11 of 13
Friedmann, E.I.; Thistle, A.B. Foreword. In Antarctic Microbiology; Friedmann, E.I., Ed.; Wiley-Liss: New York,
NY, USA, 1993; pp. ix–x.
Vincent, W.F.; Howard-Williams, C.; Broady, P.A. Microbial communities and processes in Antarctic flowing
waters. In Antarctic Microbiology; Friedmann, E.I., Ed.; Wiley-Liss: New York, NY, USA, 1993; pp. 544–569.
Zucconi, L.; Pagano, S.; Fenice, M.; Selbmann, L.; Tosi, S.; Onofri, S. Growth temperature preferences of
fungal strains from Victoria Land, Antarctica. Polar Biol. 1996, 16, 53–61. [CrossRef]
Vincent, W.F. Microbial ecosystem of Antarctica; Cambridge University Press: Cambridge, UK, 1998; p. 304.
Arcangeli, C.; Zucconi, L.; Onofri, S.; Cannistraro, S. Fluorescence study on whole Antarctic fungal spores
under enhanced UV irradiation. J. Photoch. Photobiol. B 1997, 39, 258–264. [CrossRef]
Vishniac, H.S. Psychrophilic yeasts. In Enigmatic Microorganisms and Life in Extreme Environments;
Seckbach, J., Ed.; Kluwer Academic Publishers: Dordrecht, NL, USA, 1999; pp. 315–321.
Azmi, O.R.; Seppelt, R.D. Fungi of the Windmill Islands, continental Antarctica. Effect of temperature, pH
and culture media on the growth of selected microfungi. Polar Biol. 1997, 18, 128–134. [CrossRef]
Barghini, P.; Moscatelli, D.; Garzillo, A.M.V.; Crognale, S.; Fenice, M. High production of cold-tolerant
chitinases on shrimp wastes in bench-top bioreactor by the Antarctic fungus Lecanicillium muscarium CCFEE
5003: Bioprocess optimization and characterization of two main enzymes. Enzyme Microb. Technol. 2013, 53,
331–338. [CrossRef] [PubMed]
Felse, P.A.; Panda, T. Production of microbial chitinases. A revisit. Bioprocess. Eng. 2000, 23, 127–134.
[CrossRef]
Seidl, V. Chitinases of filamentous fungi: A large group of diverse proteins with multiple physiological
functions. Fungal Biol. Rev. 2008, 22, 36–42. [CrossRef]
Ulhoa, C.J.; Peberdy, J.F. Purification and some properties of extracellular chitinase produced by Trichoderma
harzianum. Enzyme Microb. Tech. 1992, 14, 236–240. [CrossRef]
Zhu, Y.; Pan, J.; Qiu, J.; Guan, X. Isolation and characterization of a chitinase gene from entomopathogenic
fungus Verticillium lecanii. Braz. J. Microbiol. 2008, 39, 314–320.
Rocha-Pino, Z.; Vigueras, G.; Shirai, K. Production and activities of chitinases and hydrophobins from
Lecanicillium lecanii. Bioproc. Biosyst. Eng. 2011, 34, 681–686. [CrossRef] [PubMed]
Binod, P.; Sandhya, C.; Suma, P.; Szakacs, G.; Pandey, A. Fungal biosynthesis of endochitinase and chitobiase
in solid state fermentation and their application for the production of N-acetyl-D-glucosamine from colloidal
chitin. Bioresour. Technol. 2007, 98, 2742–2748. [CrossRef] [PubMed]
Fenice, M.; Selbmann, L.; di Giambattista, R.; Petruccioli, M.; Federici, F. Production of extracellular
chitinolytic activities by a strain of the Antarctic entomogenous fungus Verticillium cfr. lecanii. In Chitin
Enzymology; Muzzarelli, R.A.A., Ed.; Atec Edizioni: Grottammare, Italy, 1996; pp. 285–292.
Matsumoto, Y.; Saucedo-Castañeda, G.; Revah, S.; Shirai, K. Production of β-N-acetylhexosaminidase of
Verticillium lecanii by solid state and submerged fermentations utilizing shrimp waste silage as substrate and
inducer. Process. Biochem. 2004, 39, 665–671. [CrossRef]
Fenice, M.; Gooday, G.W. Mycoparasitic actions against fungi and oomycetes by a strain (CCFEE 5003) of the
fungus Lecanicillium muscarium isolated in Continental Antarctica. Ann. Microbiol. 2006, 56. [CrossRef]
Ramírez-Coutiño, L.; Marín-Cervantes, M.; Huerta, S.; Revah, S.; Shirai, K. Enzymatic hydrolysis of chitin
in the production of oligosaccharides using Lecanicillium fungicola chitinases. Process. Biochem. 2006, 41,
1106–1110. [CrossRef]
Barghini, P.; Esti, M.; Pasqualetti, M.; Silvi, S.; Aquilanti, A.; Fenice, M. Crude cell wall degrading enzymes,
by the Antarctic fungus Lecanicillium muscarium CCFEE 5003, inhibits the Ochratoxin-A producer Aspergillus
carbonarius on white and red grapes. J. Environ. Prot. Ecol. 2013, 14, 1673–1679.
Fenice, M.; di Giambattista, R.; Leuba, G.L.; Federici, F. Inactivation of Mucor plumbeus by the combined
action of chitinase and high hydrostatic pressure. Int. J. Food Microbiol. 1999, 52, 109–113. [CrossRef]
Malathrakis, N.E.; Kritsotaki, O. Effect of substrate, temperature and time of application on the effectiveness
of three antagonistic fungi against Botrytis cinerea. In Recent Advances in Botrytis Research; Verhoeffer, K.,
Malathrakis, N.E., Williamson, B., Eds.; Pudoc Scientific Publisher: Wageningen, The Netherlands, 1992;
pp. 187–191.
Antal, Z.; Manczinger, L.; Szakacs, G.; Tengerdy, R.P.; Ferenczy, L. Colony growth, in vitro antagonism and
secretion of extracellular enzymes in cold-tolerant strains of Trichoderma species. Mycol. Res. 2000, 104,
545–549. [CrossRef]
Molecules 2016, 21, 447
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
12 of 13
Juarez-Jimenez, B.; Rodelas, B.; Martinez-Toledo, M.V.; Gonzalez-Lopez, J.; Crognale, S.; Gallo, A.M.;
Pesciaroli, C.; Fenice, M. Production of chitinolytic enzymes by a strain (BM17) of Paenibacillus pabuli isolated
from crab shells samples collected in the East Sector of Central Tyrrhenian Sea. Int. J. Biol. Macr. 2008, 43,
27–31. [CrossRef] [PubMed]
Zare, R.; Gams, W. A revision of Verticillium sect. Prostrata. IV. The genera Lecanicillium and Simplicillium gen.
nov. Nova Hedwigia 2001, 73, 1–50.
Askary, H.; Benhamou, N.; Brodeur, J. Ultrastructural and cytochemical characterization of aphid invasion
by the hyphomycete Verticillium lecanii. J. Invertebr. Pathol. 1999, 74, 1–13. [CrossRef] [PubMed]
Marshall, R.K.; Lester, M.T.; Glare, T.R.; Christeller, J.T. The fungus, Lecanicillium muscarium, is an
entomopathogen of passionvine hopper (Scolypopa australis). N. Z. J. Crop. Hort. 2003, 31, 1–7. [CrossRef]
Cuthbertson, A.G.S.; Walters, K.F.A.; Northing, P. The susceptibility of immature stages of Bemisia tabaci to
the entomopathogenic fungus Lecanicillium muscarium on tomato and verbena foliage. Mycopathologia 2005,
159, 23–29. [CrossRef] [PubMed]
North, J.P.; Cuthbertson, A.G.; Walters, K.F. The efficacy of two entomopathogenic biocontrol agents against
adult Thrips palmi (Thysanoptera: Thripidae). J. Invertebr. Pathol. 2006, 92, 89–92. [CrossRef] [PubMed]
Askary, H.; Yarmand, H. Development of the entomopathogenic hyphomycete Lecanicillium muscarium
(Hyphomycetes: Moniliales) on various hosts. Eur. J. Entomol. 2007, 104, 67–72. [CrossRef]
Goettel, M.S.; Koike, M.; Kim, J.J.; Aiuchi, D.; Shinya, R.; Brodeur, J. Potential of Lecanicillium spp. for
management of insects, nematodes and plant diseases. J. Invertebr. Pathol. 2008, 98, 256–261. [CrossRef]
[PubMed]
Anand, R.; Tiwary, B.N. Pathogenicity of entomopathogenic fungi to eggs and larvae of Spodoptera litura,
the common cutworm. Biocontrol. Sci. Technol. 2009, 19, 919–929. [CrossRef]
Anand, R.; Prasad, B.; Tiwary, B.N. Relative susceptibility of Spodoptera litura pupae to selected
entomopathogenic fungi. BioControl 2009, 54, 85–92. [CrossRef]
De Faria, M.R.; Wraight, S.P. Mycoinsecticides and mycoacaricides: A comprehensive list with worldwide
coverage and international classification of formulation types. BiolControl 2007, 43, 237–256. [CrossRef]
Meyer, S.L.; Huettel, R.N.; Sayre, R.M. Isolation of fungi from Heterodera glycines and in vitro bioassays for
their antagonism to eggs. J. Nematol. 1990, 22, 532–537. [PubMed]
Langen, G.; Beißmann, B.; Reisener, H.J.; Kogel, K. A β-1,3-D-endo-mannanase from culture filtrates of the
hyperparasites Verticillium lecanii and Aphanocladium album that specifically lyses the germ pore plug from
uredospores of Puccinia graminis f.sp. tritici. Can. J. Bot. 1992, 70, 853–860. [CrossRef]
Askary, H.; Benhamou, N.; Brodeur, J. Ultrastructural and cytochemical investigations of the antagonistic
effect of Verticillium lecanii on cucumber powdery mildew. Phytopathology 1996, 87, 359–368. [CrossRef]
[PubMed]
Verhaar, M.A.; Hijwegen, T.; Zadoks, J.C. Glasshouse experiments on biocontrol of cucumber powdery
mildew (Sphaerotheca fuliginea) by the mycoparasites Verticillium lecanii and Sporothrix rugulosa. BiolControl
1996, 6, 353–360.
Benhamou, N.; Brodeur, I. Evidence for antibiosis and induced host defence reactions in the interaction
between Verticillium lecanii and Penicillium digitatum the causal agent of green mould. Phytopathology 2000,
90, 932–943. [CrossRef] [PubMed]
Gams, W.; Zare, R. A revision of Verticillium sect. Prostrata. III. Generic classification. Nova Hedwigia 2001,
72, 329–337.
Fenice, M.; Selbmann, L.; Zucconi, L.; Onofri, S. Production of extracellular enzymes by Antarctic fungal
strains. Polar Biol. 1997, 17, 275–280. [CrossRef]
Fenice, M.; Selbmann, L.; di Giambattista, R.; Federici, F. Chitinolytic activity at low temperature of an
Antarctic strain (A3) of Verticillium cfr. lecanii. Res. Microbiol. 1998, 149, 289–300. [CrossRef]
Moscatelli, D. Attività Chitinolitiche del Ceppo Antartico Verticillium cfr. Lecanii A3: Studio a Livello
Biochimico e Molecolare e Possibili Ricadute Applicative. Ph.D. Thesis, University of Tuscia, Viterbo, Italy,
11 July 2003.
Chet, I. Trichoderma—Application, mode of action, and potential as biocontrol agent in soilborne plant
pathogenic fungi. In Innovative Approaches to Plant Disease Control; Chet, I., Ed.; J. Wiley & Sons: New York,
NY, USA, 1997; pp. 137–160.
Molecules 2016, 21, 447
46.
47.
48.
49.
50.
51.
52.
13 of 13
Fenice, M.; Barghini, P.; Selbmann, L.; Federici, F. Combined effects of agitation and aeration on
the chitinolytic enzymes production by the Antarctic fungus Lecanicillium muscarium CCFEE 5003.
Microb. Cell Fac. 2012, 11. [CrossRef] [PubMed]
Marin-Cervantes, M.; Matsumoto, Y.; Ramírez-Coutiño, L.; Rocha-Pino, Z.; Viniegra, G.; Shirai, K. Effect of
moisture content in polyurethane foams as support for solid-substrate fermentation of Lecanicillium lecanii
on the production profiles of chitinases. Process. Biochem. 2008, 43, 24–32. [CrossRef]
Bruce, A.; Scrinivasan, U.; Staines, H.J.; Highley, T.L. Chitinase and laminarinase production in liquid culture
by Trichoderma spp. and their role in biocontrol of wood decay fungi. Int. Biodeterior. Biodegrad. 1995, 4,
337–353. [CrossRef]
Benhamou, N.; Chet, I. Hyphal interaction between Trichoderma harzianum and Rhizoctonia solani:
Ultrastructure and gold cytochemistry of the mycoparasitic process. Phytopathology 1993, 83, 1062–1071.
[CrossRef]
Inbar, J.; Menendez, A.; Chet, I. Hyphal interaction between Trichoderma harzianum and Sclerotinia sclerotiorum
and its role in biological control. Soil Biol. Biochem. 1996, 28, 757–763. [CrossRef]
Calistru, C.; McLean, M.; Berjak, P. In vitro studies on the potential for biological control of Aspergillus flavus
and Fusarium moniliforme by Trichoderma species. Mycopathologia 1997, 139, 115–121. [CrossRef] [PubMed]
Selbmann, L.; Isola, D.; Fenice, M.; Zucconi, L.; Sterflinger, K.; Onofri, S. Potential extinction of Antarctic
endemic fungal species as consequence of Global Warming. Sci. Total Environ. 2012, 438, 127–134. [CrossRef]
[PubMed]
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