Vitality and genetic fidelity of white-rot fungi mycelia following different methods of preservation

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Vitality and genetic fidelity of white-rot fungi myceliafollowing different methods of preservation

Samuele VOYRONa,*, Sophie ROUSSELb, Francoise MUNAUTb, Giovanna C. VARESEa,Marco GINEPROc, Stephan DECLERCKb, Valeria FILIPELLO MARCHISIOa

aUniversity of Turin, Department of Plant Biology, Viale P.A. Mattioli 25, 10125 Torino, ItalybMycotheque de l’Universite catholique de Louvain (BCCM/MUCL), Unite de microbiologie, Universite catholique de Louvain (UCL)

Croix du Sud 3, bte 6, B-1348 Louvain-la-Neuve, BelgiumcUniversity of Turin, Department of Analytical Chemistry, Via Pietro Giuria 5, 10125 Torino, Italy

a r t i c l e i n f o

Article history:

Received 2 September 2008

Received in revised form

22 April 2009

Accepted 12 June 2009

Available online 21 June 2009

Corresponding Editor: Teun Boekhout

Keywords:

AFLP

Basidiomycetes

Conservation

Cryopreservation

Lyophilisation

* Corresponding author. Dipartimento di BTel.: þ39 11 6705964; fax: þ39 11 6705962.

E-mail addresses: [email protected]@unito.it (G. C. Varese), [email protected] (V. Filipello March0953-7562/$ – see front matter ª 2009 The Bdoi:10.1016/j.mycres.2009.06.006

a b s t r a c t

Basidiomycetes present specific problems with regard to their preservation, because most

of them do not form resistant propagules in culture but exist only as mycelium. Usually

these fungi can only be preserved by serial transfer on agar (labour-intensive procedures

that can increase the danger of variation or loss of physiological or morphological

features), or cryopreserved in liquid nitrogen (expensive). Cryopreservation at �80 �C and

lyophilisation could be good alternatives.

In this work we set up and tested six protocols of cryopreservation at �80 �C, and 12 pro-

tocols of lyophilisation on 15 isolates of white-rot fungi (WRF) belonging to 10 species. The

tested protocols were mainly characterized by the use of different growth media, protec-

tants, time and number of perfusion with protectants and finally by the typology and origin

of the samples to be cryopreserved (mycelium/agar plug, whole colony) or to lyophilise

(mycelium/agar plug, mycelium fragment, whole colony). Cryopreservation and lyophilisa-

tion outcomes were checked, at morphological (macro- and microscopic features), physio-

logical (growth rate and laccase, Mn-independent and Mn-dependent peroxidases

activities) and genetic level (Amplified Fragment Length Polymorphisms analysis – AFLP).

Vitality of all fungi was successfully preserved by all cryopreservation protocols at

�80 �C, and by two lyophilisation methods. Our results showed that cryopreservation at

�80 �C did not produce morphological changes in any isolate, while two isolates were af-

fected by lyophilisation. None of the physiological features were lost, even though growth

rate and enzyme activities were somehow influenced by all preservation methods. AFLP

analysis showed that only the two isolates that varied in their morphology after lyophilisa-

tion produced a different DNA fingerprint pattern in comparison with that obtained before

lyophilisation. These findings provide evidence that cryopreservation at �80 �C and lyophi-

lisation are suitable alternatives to liquid nitrogen cryopreservation for preservation of

some WRF strains.

ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

iologia Vegetale, Viale Mattioli 25, I-10125 Turin, Italy.

.it (S. Voyron), [email protected] (S. Roussel), [email protected] (F. Munaut),[email protected] (M. Ginepro), [email protected] (S. Declerck), valeria.isio)ritish Mycological Society. Published by Elsevier Ltd. All rights reserved.

1028 S. Voyron et al.

IntroductionWhite-rot fungi (WRF) are the only organisms able to min-

The increasing development of biotechnological applications

using basidiomycetes requires the set up of ex-situ conserva-

tion protocols, in order to preserve, for a long time, not only

their vitality but also their physiological, morphological and

genetic features. Actually, damage to phenotypic and/or geno-

typic features originated by preservation protocols could lead

to a decrease of their biotechnological value. The fungal col-

lection of the University of Turin, the Mycotheca Universitatis

Taurinensis (MUT), maintains over 600 isolates of basidiomy-

cetes belonging to 229 species. Preservation of basidiomycetes

is rather difficult because they are often characterized by the

inability to form any resistant propagules in pure cultures.

Usually basidiomycetes can only be preserved by serial trans-

fer on agar with or without the addition of mineral oil, or on

agar medium under sterile distilled water, or cryopreserved

in liquid nitrogen. The preservation as actively growing cul-

tures (sub-culturing) or slackened (under sterile distilled wa-

ter or mineral oil) can keep isolates alive for a long time, but

many researchers’ experiments have shown that morpholog-

ical or physiological alterations are directly proportional to the

duration of preservation (Kitamoto et al. 2002; Lopez-Martınez

et al. 1999; Sharma & Smith 1999; Smith 1988, 1991; Smith &

Onions 1983, 1994). Storage in liquid nitrogen has been consid-

ered as the best and most widely applicable preservation tech-

nique available for filamentous fungi (Croan et al. 1999; Douds

& Schenck 1990; Espinel-Ingroff et al. 2004; Hoffmann 1989;

Holden & Smith 1992; Homolka et al. 2001, 2003, 2006,

2007a,b; Hwang 1960, 1966; Mata & Perez-Merlo 2003; Singh

et al. 2004a; Smith 1983, 1993, 1998; Stalpers et al. 1987; Stoy-

chev et al. 1998). The main advantage of this method is that

it preserves genomic and phenotypic features of strains and

protects cultures from contamination. Nevertheless, liquid ni-

trogen cryopreservation is very expensive, and the storage

vessels must be kept in a well-ventilated room, as the con-

stant evaporation of the nitrogen gas could displace the air

and suffocate the workers.

Cryopreservation at�80 �C and lyophilisation could be good

alternatives to overcome these problems. However, few data

are available about non-sporulating fungi cryopreservation at

�80 �C (Ito 1991; Ito & Nakagiri 1996; Kitamoto et al. 2002).

The preservation by lyophilisation was first extensively

used with fungal cultures by Raper & Alexander (1945). Long-

term preservation is the main advantage of this method;

lyophilised cultures in ampoules sealed under vacuum can

be easily stored in a small place, require no maintenance,

and can be shipped without special requirements. Moreover,

isolates are protected from infection and infestation. Nowa-

days, lyophilisation is a commonly used method to preserve

sporulating fungi. Only little evidences that lyophilisation

can be applied for non-sporulating basidiomycetes has/

have?? been provided. Tan & Stalpers (1991) demonstrated

the possibility to freeze dry mycelium of Schizophyllum com-

mune. Sundari & Adholeya (1999, 2000a,b) successfully lyophi-

lised the vegetative mycelium of Laccaria fraterna. Croan (2000)

lyophilised some tropical wood-inhabiting basidiomycetes,

and Singh et al. (2004b) set up a lyophilisation method for

saprotrophic edible mushrooms.

eralise lignin to carbon dioxide and water, and produce a com-

plex pool of enzymes including laccase, peroxidase,

cellulases, and other enzymes involved in radical, reactive ox-

ygen species, and hydrogen peroxide formation (Ralph &

Catcheside 2002). These enzyme systems transform not only

lignin, but also a wide range of chemicals that are relatively

long-lived in the environment by virtue of their high molecu-

lar weight, insolubility, chemical irregularity, thermodynamic

stability or recent origin precluding the evolution of specific

microbial decay mechanisms (Ralph & Catcheside 2002). Cat-

abolic versatility appears to be a generic feature of WRF and

makes them very useful for their application in novel biotech-

nological applications.

The first objective of this study was to test different protocols

of lyophilisation and of cryopreservation at �80 �C on different

species of WRF, and to check, after revival, not only the isolates

vitality, but also some morpho-physiological features, such as

macro-and microscopicmorphology, growthand enzymaticac-

tivities. Based on these results, the second objective was to se-

lect one protocol of cryopreservation at �80 �C and at least one

of lyophilisation, and to evaluate the presence of genetic varia-

tions by means of the Amplified Fragment Length Polymor-

phisms technique (AFLP) (Vos et al. 1995). The AFLP is a useful

technique for the control of the outcomes of preservation

methodsbecauseanygenotypicchange,whichcouldhaveaper-

manent and heritable effect on the species, will be pointed out.

Materials and methods

Organisms

The WRF isolates examined and their origin are listed in

Table 1. All the isolates used in this work are deposited at

MUT. They were previously isolated from carpophore at

MUT laboratories and preserved, as actively growing cultures,

for two years on a home made agar medium (PIAM) at 4 �C.

Species identification was based upon morphological fea-

tures. WRF maintained during study time as actively growing

cultures were used as controls.

Culture media

Growth medium PIAM consists of 20 g malt extract supple-

mented by vitamins (in 20 g: vitamin C 16 mg; vitamin PP

4 mg; riboflavin 0.6 mg; thiamine 0.4 mg; vitamin B6 0.5 mg; vi-

tamin D 0.002 mg, Vecchi & C. PIAM, Italy), 2 g peptone from

casein acid digest, 20 g glucose, 0.2 g KH2PO4, 16 g agar-agar,

in trace FeCl3, per 1000 ml distilled water. Growth medium

PIAM broth is medium PIAM without agar. Growth medium

PIAM gly consists of PIAM supplemented with 5 % (w/v) glyc-

erol. Growth medium MPGT consists of 30 g malt extract, 5 g

peptone from casein acid digest, 2.5 g glucose, 20 g trehalose,

18 g agar-agar, per 1000 ml distilled water. Growth medium

MPGT broth is medium MPGT without agar. Growth medium

MEA consists of 20 g malt extract, 2 g peptone from casein

acid digest, 20 g glucose, and 20 g agar-agar, per 1000 ml dis-

tilled water. Sterile agar media were plated in 60 mm diameter

Table 1 – Species and origin of the white-rot fungi strains used (s [ saprotroph; p [ parasite)

Species MUT N� Origin Trophism

Coriolopsis gallica 3379 Dead wood, Parco della Mandria (TO) Italy s

C. gallica 3380 Hazelnut dead wood, Parco della Mandria (TO) Italy s

C. gallica 3382 Dead wood, Parco della Mandria (TO) Italy s

Daedaleopsis confragosa var. confragosa 3483 Hornbeam branch, Parco della Mandria (TO) Italy s

Ganoderma adspersum 3426 On red oak, Parco della Mandria (TO) Italy p

G. adspersum 3427 On red oak, Parco della Mandria (TO) Italy p

Lenzites betulina 3368 On red oak, Parco della Mandria (TO) Italy s

Plicaturopsis crispa 3394 Dead wood, Parco della Mandria (TO) Italy s

P. crispa 3496 Alder dead wood, Parco della Mandria (TO) Italy s

Schizophyllum commune 3392 Hazelnut dead wood Parco della Mandria (TO) Italy s

Schizopora paradoxa 3389 Oak dead wood Parco della Mandria (TO) Italy s

S. paradoxa 3390 Dead wood, Parco della Mandria (TO) Italy s

Trametes gibbosa 3364 Hornbeam branch, Parco della Mandria (TO) Italy s

Trametes pubescens 2400 Decaying trunk of poplar, Franosa (CN) Italy s

Trametes versicolor 3374 Oak dead trunk, Parco della Mandria (TO) Italy s

MUT¼Mycotheca Universitatis Taurinenesis.

Vitality and genetic fidelity of white-rot fungi following preservation 1029

Petri dishes. Growth medium PIAM was also plated in 150 mm

diameter Petri dishes for growth rate evaluation.

Preservation methods

The tested preservation methods were chosen on the basis of

literature data, unpublished data from previous experiments

carried out at MUT laboratories, and considering our facilities.

The 6 cryopreservation protocols differed in the use of dif-

ferent growth media at the start of preservation procedures

(MPGT agar or PIAM gly), protectants (glycerol or trehalose),

time and temperature of perfusion with protectants, and by

the typology and origin of the samples to be cryopreserved (my-

celium/agar plug or whole colony). The 12 lyophilisation proto-

cols differed in the use of four different growth media at the

start of preservation procedures (PIAM agar or broth, MPGT

agar or broth), two protectants (suspending medium contain-

ing skimmed milk and trehalose or skimmed milk and myo-

inositol), time and number of perfusion with protectants, and

by the typology and origin of the samples to be lyophilised

(mycelium/agar plug, mycelium fragment, or whole colony).

Cryopreservation protocols

Cryopreservation group C1, protocols PC1 and MC1(Hoffmann 1989), modified as followsIsolates were inoculated on PIAM gly agar (PC1) or MPGT agar

(MC1) and incubated at 24 �C in the dark. After 1–3 weeks of

growth, well-developed colonies were flooded with a 10 %

(w/v) glycerol H2O solution sterilized by autoclaving (20 min

at 121 �C). The incubation time was chosen on the basis of

the growth rate of each tested isolate. Flooded colonies were

incubated for 1 h at room temperature to allow mycelium ad-

aptation to the cryoprotective. A sterile straw, open at both

ends, was then used to punch the mycelium and the underly-

ing agar (mycelium/agar plug) from the margin of an actively

growing colony. After the collection of five mycelium/agar

plugs, the straw was cut by a sterile straw cutter (Straw-Cut,

Cryo Bio Systems, IMV Technologies, France) and sealed

(SYMS Sealing System, Cryo Bio Systems, IMV Technologies,

France) at one end. Three straws were then aseptically trans-

ferred into a 2 ml a sterile cryotube (Nalgene, USA).

To obtain a freezing rate that is close to the theoretical op-

timum of 1 �C min�1, the cryotubes placed in a freezing con-

tainer filled with isopropyl alcohol (5100 Cryo ‘‘Mr. Frosty’’

Nalgene) were transferred for 2 h into a mechanical deep

freezer at �80 �C (Sanyo mod. MDFU6086S). Cryotubes were

then stored at �80 �C.

For revival, straws were surface sterilized for 30 s by im-

mersion in ethanol 70 % (v/v), opened with sterile scissors

and the frozen mycelium/agar plugs were thawed directly

on PIAM, and then incubated at 24 �C in the dark.

Cryopreservation group C2, protocols PTC2, PGC2, MTC2,MGC2 (Smith & Onions 1994), modified as followsIsolates were inoculated on PIAM gly agar (PTC2 or PGC2) or

MPGT agar (MTC2 or MGC2) and incubated at 24 �C in the

dark until measurable growth occurred. 2 ml sterile cryotubes

(Nalgene) were filled up to 1/3 of their length with PIAM agar or

MPGT agar. One mycelium/agar plug picked up from the mar-

gin of an actively growing colony by means of a sterile cap-

punch in brass with a diameter of 5 mm was transferred

into cryotube.

Colonies in cryotubes were then incubated at 24 �C for 1–3

weeks in the dark. Well-developed colonies were then flooded

with a 10 % (w/v) glycerol H2O solution sterilized by autoclav-

ing (20 min at 121 �C) for protocols PGC2 and MGC2, or with

a 10 % (w/v) D (þ) trehalose H2O solution sterilized by autoclav-

ing (20 min at 121 �C) for protocols PTC2 and MTC2. The incu-

bation time was chosen on the basis of the growth rate of each

tested isolate. Flooded colony were then incubated for 4 h at

4 �C. Samples were frozen as described for the C1 protocol.

For revival, cryotubes were transferred into a water bath at

37 �C until all ice melted. Fungi were then inoculated on PIAM,

and incubated at 24 �C in the dark.

Lyophilisation protocols

Lyophilisation group L1, protocols PTL1, PML1, MTL1, MML1(Croan 2000), modified as follows

1030 S. Voyron et al.

Isolates were inoculated on PIAM agar (PTL1 and PML1) or MPGT

agar (MTL1 and MML1) and incubated at 24 �C in the dark until

measurable growth occurred. By means of a sterile cappunch in

brass, with a diameter of 5 mm, 3 mycelium/agar plugs were

collected from the margin of an actively growing colony. The

agar plugs were then transferred into cotton plugged 2 ml ster-

ile constricted glass ampoules (Wheaton, USA). For each tested

isolate, 5 lyophilisation vials were prepared. Mycelium/agar

plugs were then incubated at 24 �C in the dark. After 1–2 weeks

of growth, the mycelium/agar plugs were flooded with two dif-

ferent sterile lyophilisation suspending media consisting of

0.6 ml of a 10 % (w/v) skimmed milk, 10 % (w/v) D (þ) trehalose

H2O solution for PTL1 and MTL1, or of 0.6 ml of a 10 % (w/v)

skimmed milk, and 10 % (w/v) myo-inositol H2O solution for

PML1 and MML1. The incubation time was chosen on the basis

of the growth rate of each tested isolate.

Flooded colonies were incubated overnight at 4 �C and

lyophilised using a shelf freeze-drying model LIO10P (5-Pascal,

Italy). Samples were cooled to �24 �C with a cooling rate of

1 �C min�1, and the lyophilisation chamber was then evacuated.

After the evacuation the temperature was retained at�35 �C for

3 h, and then raised to þ10 �C at a rate of 0.08 �C min�1. Then

lyophilisation vials were plugged, under vacuum, by a rubber

bung and finally sealed using a cross-fire burner.

Lyophilisation group L2, protocols PTL2, PML2, MTL2, MML2(Croan 2000), modified as followsMycelium/agar plugs picked up from the margin of an actively

growing colony on PIAM (PTL2 and PML2) agar or MPGT agar

(MTL2 and MML2) were inoculated into 25 ml PIAM broth

(PTL2 and PML2) or MPGT broth (MTL2 and MML2) in

a 100 ml Erlenmeyer flask and incubated at 24 �C in the dark,

until the mycelium covered the entire surface of the medium.

The biomass was then torn into fragments of approximately

0.5 cm2. The biomass fragments were then rinsed with a sterile

0.005 % Tween 20 H2O solution. Three mycelial fragments

were then transferred into cotton plugged 2 ml sterile con-

stricted glass ampoules (Wheaton). For each tested isolate, 5

lyophilisation vials were prepared. Lyophilisation vials were

flooded with each of the two sterile lyophilisation suspending

media, consisting of 0.4 ml of a 10 % (w/v) skimmed milk and

10 % (w/v) D (þ) trehalose H2O solution for PTL2 and MTL2, or of

0.4 ml of a 10 % (w/v) skimmed milk and 10 % (w/v) myo-

inositol H2O solution, for PML2 and MML2.

Biomasses were then incubated in the lyophilisation sus-

pending medium at 24 �C in the dark. After 1–3 weeks of incuba-

tion, colonies were then flooded again with the lyophilisation

suspendingmediumconsistingof0.3 ml of a 10% (w/v) skimmed

milk and 10 % (w/v) D (þ) trehalose H2O solution for PTL2 and

MTL2, or of 0.3 ml of a 10 % (w/v) skimmed milk and 10 % (w/v)

myo-inositol H2O solution for PML2 and MML2. The incubation

time was chosen on the basis of the growth rate of each tested

isolate. Flooded colonies were incubated overnight at 4 �C and

lyophilised according to the method described for the L1 group.

Lyophilisation group L3, protocols PTL3, PML3, MTL3, MML3Isolates were inoculated on PIAM agar (PTL3 and PML3) or on

MPGT agar (MTL3 and MML3) and incubated at 24 �C in the

dark until measurable growth occurred. Cotton plugged 2 ml

sterile constricted glass ampoules (Wheaton) were filled up

to 1/3 of their length with PIAM agar or MPGT agar. One myce-

lium/agar plug picked up from the margin of an actively grow-

ing colony, by means of a sterile cappunch in brass with

a diameter of 5 mm, was transferred in the ampoules contain-

ing PIAM agar (PTL3 and PML3) or MPGT agar (MTL3 and

MML3). For each tested isolate, 5 lyophilisation vials were pre-

pared. The vials were incubated at 24 �C for 1–2 weeks. The in-

cubation time was chosen on the basis of the growth rate of

each tested isolate. Well-developed colonies were then

flooded with the lyophilisation suspending medium, consist-

ing of 0.3 ml of a 10 % (w/v) skimmed milk, 10 % (w/v) D (þ) tre-

halose H2O solution for PTL3 and MTL3, or of 0.3 ml of a 10 %

(w/v) skimmed milk, 10 % (w/v) myo-inositol H2O solution

for PML3 and MML3. Flooded colonies were incubated over-

night at 4 �C and lyophilised according to the method de-

scribed for the L1 group.

Evaluation of residual moisture content and glass transitiontemperature of dried material

To establish the residual moisture content of the dried mate-

rial, the water content of 5 lyophilised samples, randomly

chosen from different lyophilisation cycles, was determined

by Karl Fischer method following the Metrohm KF Application

Note AN-K-004.

To establish the glass transition temperature (Tg) of dried

material, 5 lyophilised samples, randomly chosen from differ-

ent lyophilisation cycles, were analysed by differential scan-

ning calorimetry (DSC – N2 flux of 50 cm3 min�1, heating rate

of 20 �C min�1) using a Differential Scanning Calorimeter

Q200 (TA Instruments, USA). The resulting thermal scans

were analysed by the TA Universal Analysis Software 2000

(TA Instruments).

Vitality tests

For revival, lyophilised isolates were flooded with sterile dis-

tilled water and maintained for 1 h at room temperature. My-

celium/agar plugs from protocol L1, mycelial fragments from

protocol L2, a part of a colony from protocol L3 were then in-

oculated on PIAM agar and incubated at 24 �C in the dark.

For all cryopreservation and lyophilisation protocols vitality

was tested after one month of preservation.

Morphological and physiological analyses

Morphological parametersThe morphological analysis was carried out on control cul-

tures and on those arising from the vitality tests. The analysis

was addressed to check the eventual presence of morpholog-

ical variations after preservation and not to describe the spe-

cies used in this work. For this reason we have considered

only a limited set of features, following the indications of

Stalpers (1978). The colour codes refer to the colour identifica-

tion chart of Rayner (1970).

The following macroscopic features have been considered:

colony colour (uncoloured, white, cream, yellowish, brown-

ish, orange, pink); reverse colour; texture of mycelium

(absent, downy, farinaceous, granular, silky, cottony, woolly,

Vitality and genetic fidelity of white-rot fungi following preservation 1031

floccose, plumose, pellicular or subfelty, felty, velvety, crus-

tose, lacunose, zonate). At the microscopical level, the follow-

ing characteristics have been observed using a Leica DM

4500B microscope at magnification 250, 400 and 630:

presence/absence of clamp connections; presence/absence

of hyphal vacuolization. The experiment was repeated for

three generations after revival.

Growth rateFrom the margin of well-developed control cultures and of

those arosen from the vitality tests (7–15 d old, depending on

strain), a 5 mm diameter mycelium/agar plug was picked up

by means of a sterile cappunch in brass and inoculated in

150 mm diameter plates containing PIAM agar, and incubated

at 24 �C in the dark. For each tested and control isolate, six rep-

lications were carried out. Each colony was photographed ev-

ery two days starting from the third day of incubation. These

data were used to build the growth curves of each isolate (con-

trol and treated). The end of experiment (the growth end point)

was set up at 15 d or when the colony was grown all over the

plate. The growth was determined by measuring the area

(cm2) occupied by colonies with the image analysis software

IMAGEJ 1.36b (http://www.ansci.wisc.edu/equine/parrish/

index.html). In order to evaluate difference in growth between

treated and control isolates, values at growth end point (mean

of the 6 replications) were compared. Significant differences

between treated and control samples were assessed by

Mann–Whitney test (SPSS 13.0 software, SPSS Italy) ( p� 0.05).

Enzymatic activityThe starting inoculum was standardized by scrapping 35 mg

of mycelium from the margin of control cultures, and of those

arising from the vitality tests. The mycelium was then homog-

enized for 30 s in 500 ml of PIAM broth, and 25 ml were inocu-

lated in 24 Multi-well plate containing 1.5 ml/well of PIAM

broth. For each trial, 5 replications were carried out. Isolates

were incubated at 24 �C in the dark. The incubation time for

each sample was chosen on the basis of the growth end point.

Enzymes tested – Extracellular Laccase (Lac), Peroxidase Mn-

independent (MiP) and Mn-dependent (MnP) activities were

measured by standard spectrophotometrical methods, using

an Ultrospec 3300 Pro (Amersham Biosciences, USA).

Lac activity was measured by ABTS [2,20-azino-bis(3-ethyl-

benzo-thiazoline-6-sulfonic acid)diammonium salt] oxidation

at l¼ 420 nm, using a molar extinction coefficient (E420) of

36 000 M�1 cm�1 (Niku-Paavola et al. 1988). MiP and MnP activ-

ities were measured by DMAB/MBTH [3-(dimethylamino)

benzoic acid/3-methyl-2-benzothiazolinone hydrazone hy-

drochloride] oxidation at l¼ 590 nm, using a molar extinction

coefficient (E590) of 32 900 M�1 cm�1 (Vyas et al. 1994). Values

obtained from controls, lyophilised and cryopreserved fungi

were compared. The presence of significant differences was

assessed by Mann–Whitney test (SPSS 13.0 software, SPSS

Italy) ( p� 0.05).

Principal Coordinate Analysis (PCoA)

A binary matrix was built to assess the optimal preservation

protocols on the basis of the complete set of data obtained

from both control and treated isolates: vitality, growth end

point (6 replicates), morphology (3 replicates) and enzymatic

activity (5 replicates for each tested enzyme). We assigned

the arbitrary value 1 to all control data and data of all treated

isolate that were not significantly different from the control

data, while the arbitrary value 0 was assigned to data of all sig-

nificantly different treated isolates. This matrix was then ana-

lysed using the statistic analysis software Syntax 2000 (Exter

Software, USA) using the PCoA method and the Jaccard’s

coefficient for binary data.

Genetic fidelity

The analysis was applied to a restricted number of preserva-

tion protocols chosen on the basis of the PCoA analysis.

DNA extractionRevived and control isolates were inoculated on MEA and in-

cubated at 24 �C in the dark for 1–2 weeks. From each sample,

300 mg of fresh mycelium was collected and crushed using

1 mm zirconia/silica beads (Bio-Spec Inc., USA) by means of

MagNA Lyser (Roche, Switzerland) at a speed of 6000 rpm for

30 s. 700 ml of lysis solution (50 mM Tris–HCl pH 7.2; 50 mM

EDTA; 3 % (w/v) SDS) and 7 ml of b-mercaptoethanol were

added to each crushed sample. Samples were incubated at

65 �C for 1 h and centrifuged at 13 000 rpm for 15 min (centri-

fuge Eppendorf Mod. 5417 R, Eppendorf, Germany). The super-

natants were transferred into 1.5 ml tubes and 50 mg of RNAse

A (Invitek GmbH, Germany) were added to samples, which

were then incubated for 30 min at 50 �C. 700 ml of a phenol/iso-

amyl alcohol/chloroform (25:1:24) mixture were added to sam-

ples that were then shaken and centrifuged at 13 000 rpm for

15 min. The supernatants were transferred into test tubes,

and 700 ml of isoamilic alcohol/chloroform (1:24) solution

were added. Samples were then shaken and centrifuged at

13 000 rpm for 15 min. The supernatants were transferred

into 1.5 ml tubes and 800 ml of absolute ethanol were added

to each tube. Samples were then incubated at �80 �C for 1 h

and centrifuged at 13 000 rpm for 20 min at 4 �C. The superna-

tants were discarded, the pellets were air dried and dissolved

in 100 ml of sterile distilled water at 55 �C. The DNA was quan-

tified by means of a spectrophotometer (BioPhotometer,

Eppendorf).

AFLP procedureRestriction and ligation of adapters – Adapter and primer se-

quences used for the AFLP analysis are listed in Table 2. Re-

striction and ligation steps were performed with the AFLP

Core Reagent kit (Invitrogen, USA) according to the manufac-

turer’s instructions with only one modification: restriction

was performed in a total volume of 10 ml instead of 25 ml. A to-

tal of 250 ng of genomic DNA was digested with 2 ml of the mix-

ture of EcoRI- and MseI-endonucleases and 1 ml of 5� buffer

for 2 h at 37 �C. The samples were incubated for 15 min at

70 �C then placed on ice. The resulting fragments were ligated

by means of 24 ml of adapter-ligation solution and 1 ml of

T4DNA ligase. Samples were then incubated for 1 h at 20 �C.

The restriction–ligation mixture was diluted twenty fold

with sterile water, and then amplified by PCR.

Pre-selective PCR – Pre-selective PCR was performedusing the

core sequences, i.e. MseI site primer (M) and EcoRI site primers

Table 2 – Sequences of the adapters and primers used forthe AFLP analysis

Adapter, primer andprimer code

Sequence

Adapter EcoRI 50-CTC GTA GAC TGC GTA CC-30

50-AAT TGG TAC GCA GTC TAC-30

Adapter MseI 50-GAC GAT GAG TCC TGA G-30

50-TAC TCA GGA CTC AT-30

EcoRI (E)a 50-GACTGCGTACCAATTC-30

MseI (M)a,b 50-GATGAGTCCTGAGTAA-30

EcoRI (E-D4)b,c 50-(D4)-GACTGCGTACCAATTC-30

EcoRI-C (EC-D4)b,c 50-(D4)-GACTGCGTACCAATTCC-30

EcoRI-G (EG-D4)b,c 50-(D4)-GACTGCGTACCAATTCG-30

MseI-C (MC)b 50-GATGAGTCCTGAGTAAC-30

MseI-G (MG)b 50-GATGAGTCCTGAGTAAG-30

a Primers used for the pre-selective PCR.

b Primers used for the selective PCR.

c D4: D4 WellRED fluorescent dye, Beckman Coulter.

1032 S. Voyron et al.

(E). The PCR amplification was performed in a final volume of

25 ml with 0.5 ml of a solution 10 mM of each EcoRI- and MseI-

core sequence and 15 ml of Amplification Core Mix from the

AFLP� Microbial Fingerprinting kit (Applied Biosystems, USA)

under the following conditions: 94 �C for 2 min; 94 �C for 20 s,

56 �C for 30 s, 72 �C for 2 min (30 cycles); 72 �C for 2 min; 60 �C

for 30 min. The reaction was then maintained at 4 �C.

Selective PCR – Four combinations between D4 labelled EcoRI

primers (D4 WellRED fluorescent dye, Sigma-Proligo, Beckman

Coulter license, USA) and three MS primers were tested: E-D4/

MC; E-D4/MG; EC-D4/M; EG-D4/M. The other 4 possible combi-

nations (EC-D4/MC; EC-D4/MG; EG-D4/MC; EG-D4/MG) were

discarded due to the few number of bands reported.

The selective PCR amplification was performed in a final

volume of 10 ml. The final mix contained 1.5 ml of the pre-am-

plification mix, 0.6 ml of 5 mM MseI primer solution, 0.4 ml of

5 mM EcoRI primer solution D4 fluorescent labelled and 7.5 ml

of Amplification Core Mix AFLP� Microbial Fingerprinting kit

(Applied Biosystems).

The touchdown PCR amplification took place under the fol-

lowing conditions: 94 �C for 2 min; 94 �C for 20 s, 66 �C for 30 s,

72 �C for 2 min (10 cycles); 94 �C for 20 s, 56 �C for 30 s, 72 �C for

2 min (30 cycles) then 60 �C for 10 min. The reaction was then

maintained at 4 �C.

Capillary electrophoresis and data analysis – Capillary electro-

phoresis was performed on the CEQ� 2000 Genetic Analysis

System (Beckman Coulter). Two microlitres of the PCR prod-

ucts were combined with 30 ml of SLS loading mix (Sample

Loading Solution, Beckman Coulter) and 0.5 ml of the CEQ

DNA size standard kit-600 (Beckman Coulter). Samples were

run on the CEQ� 2000 Genetic Analysis System under the stan-

dard method FRAG-2: capillary temperature of 35 �C, denatur-

ing temperature of 90 �C for 2 min, injection voltage of 2.0 kV

for 1 min and separation voltage of 6.0 kV for 60 min. The total

running time for one row of eight samples was 85 min.

AFLP analysis – The size and identity of the amplified frag-

ments were determined by the Fragment Analysis Module of

the CEQ 2000 (Beckman Coulter). Only amplified fragments

with size ranging from 60 to 500 base pairs were scored since

the software cannot size accurately bands outside this range.

As recommended by manufacturers, a minimum fluorescence

threshold value of 500 was chosen, but lower peaks were

sometimes considered, especially for high molecular weight

fragments, when their resolution was comparable to those of

ladder fragments having similar size. The presence or absence

of polymorphic DNA fragments was given in binary characters

(1 or 0). With the four AFLP primer pairs we generated a multi-

locus DNA fingerprint for each control and treated isolate. The

AFLP fingerprints of treated isolates were then compared for

differences (presence/absence of fragments) with those

obtained with control isolates. The difference rate was then

calculated as the total number of differences per profile di-

vided by the total number of fragments per control profile

and expressed as a percentage value (Bonin et al. 2004).

AFLP Technical error assessment – The technical error was

evaluated following Bonin et al. (2004) and Pompanon et al.

(2005). Therefore, the complete AFLP procedure was repeated

for two control isolates (Schizopora paradoxa MUT 3389 and Pli-

caturopsis crispa MUT 3496) using two different restriction–liga-

tion products of the same genomic DNA. One profile was

randomly chosen as the control profile and then the dupli-

cated AFLP profile was compared to the control one. The tech-

nical error rate was then calculated as the total number of

differences per profile divided by the total number of frag-

ments per control profile and expressed as a percentage value.

Results

Vitality – After one month of storage at �80 �C, all tested WRF

have survived under the six cryopreservation conditions, irre-

spective to the cryoprotective (glycerol or trehalose), the

growth media, the perfusion time and temperature, and the

thawing temperature.

All tested WRF survived lyophilisation conditions MTL2

and PTL2, good vitality results were obtained also with proto-

cols of group L3, while only 4 out of 15 isolates survived proto-

col of group L1 (Coriolopsis gallica MUT 3380; Schizopora

paradoxa MUT 3389 and 3390, Trametes pubescens MUT 2400)

(Table 3). Due to the low vitality reported, all variants of the

protocol group L1 were discarded, and the subsequent analy-

ses were carried out only on the six cryopreservation methods

and on lyophilisation protocols of group L2 and L3.

The analysis of the residual moisture content by Karl

Fisher analysis showed that the water content of lyophilised

samples ranged from 2.02 % to 2.89 % with a mean of 2.55 %.

The DSC analysis showed that Tg of lyophilised samples

ranged from 59.89 to 67.71 �C with a mean of 64.45 �C.

Morphology – Cryopreservation at �80 �C did not produce

morphological changes in any isolate. Ganoderma adspersum

MUT 3426 and MUT 3427 were affected by lyophilisation. Mor-

phological modifications were followed in three generations

after revival. In the isolate G. adspersum MUT 3426 we found

macro- and micromorphological modifications while in the

isolate G. adspersum MUT 3427 only micromorphological mod-

ifications. After lyophilisation using protocols MTL2 and PTL2,

G. adspersum MUT 3426 at the age of 12–15 d showed a colony

colour lightly orange (6F), with some darker areas (11 SIENNA;

9H), while the colony colour of the control isolate was pure

Table 4 – Percentages of isolates that display a significantdifference from control values, for growth end point,Laccase (Lac), Peroxidase Mn-independent (MiP) andPeroxidase Mn-dependent (MnP) activity

Treatmenta Growthb Enzymatic activitiesc

Lacd Mipe MnPf

þ � þ � þ � þ �

MTL2 40 7 13 13 7 13 13 0

MML2 33 11 33 22 0 22 0 0

PTL2 40 7 27 13 0 13 13 0

PML2 40 20 10 40 10 20 0 0

MTL3 33 0 25 25 0 8 8 0

MML3 27 0 36 27 0 9 9 0

PTL3 10 0 30 10 0 20 10 0

PML3 29 0 29 43 0 14 0 0

MC1 20 27 27 13 20 13 7 7

PC1 13 20 20 13 13 0 7 7

MTC2 27 20 20 13 13 7 7 13

MGC2 33 20 13 7 20 7 7 0

PTC2 7 27 13 20 0 13 0 0

PGC2 20 27 13 40 7 13 0 13

a Variants of the lyophilisation protocols L2 and L3; variants of the

cryopreservation protocols C1 and C2.

b Percentage of isolates that display a significant decrease of

growth rate; þ: percentage of isolates that display a significant

increase of growth rate.

c Percentage of isolates that display a significant decrease enzy-

matic activity; þ: percentage of isolates that display a significant

increase of enzymatic activity.

d Laccase activity.

e Peroxidase Mn-independent activity.

f Peroxidase Mn-dependent activity.

Table 3 – Vitality of the 15 white-rot isolates after 1 m of preservation by 12 different lyophilisation protocols

Species MUT N� L1a L2a L3a

MTL1b MML1b PTL1b PML1b MTL2b MML2b PTL2b PML2b MTL3b MML3b PTL3b PML3b

C. gallica 3379 D D D D A A A A A A A A

C. gallica 3380 A A A A A A A y A A A A

C. gallica 3382 D D D D A A A A A A A A

D. confragosa var. confragosa 3483 D D D D A D A D D A D D

G. adspersum 3426 D D D D A D A D D D D D

G. adspersum 3427 D D D D A D A D A A A D

L. betulinus 3368 D D D D A A A A A A D D

P. crispa 3394 D D D D A D A A A y A A

P. crispa 3496 D D D D A D A A A A D D

S. commune 3392 D D D D A A A A A A A A

S. paradoxa 3389 A A A A A D A A D D A D

S. paradoxa 3390 A A A D A A A A A D D D

T. gibbosa 3364 D D D D A A A A A A A A

T. pubescens 2400 A A A A A A A A A A A A

T. versicolor 3374 D D D D A A A A A A A A

Vitality (%) 27 27 27 20 100 60 100 73 80 73 67 53

MUT¼Mycotheca Universitatis Taurinenesis.

‘‘A’’¼ alive; ‘‘D’’¼ death.

a Lyophilisation protocols.

b Variants of the lyophilisation protocols.

Vitality and genetic fidelity of white-rot fungi following preservation 1033

white. The control started to be lightly orange (6F), with darker

areas (11 SIENNA; 9H), only after 21 d of growth. In treated iso-

lates, the aerial mycelium was partially submerged with many

aerial woolly spots. By contrast, the mycelium of control isolate

was homogeneous and cottony/crustose. Under the micro-

scope, treated samples showed hyphae densely vacuolized al-

ready after 7 d of growth, while control hyphae were

vacuolized only after 21 d of growth. After lyophilisation by

PTL2, PTL3, MTL3 and MML3, G. adspersum MUT 3427 showed

densely vacuolated hyphae already after 7 d of growth, while

the control ones were strongly vacuolated only after 21 d.

Growth – The isolate growth curves are not affected by the

long-term preservation methods; all revived cryopreserved

and lyophilised isolates exhibited a regular exponential

growth (data not shown). The analysis of growth rate as fre-

quency of significant differences from control values shows

that lyophilisation mostly stimulates the growth, while cryo-

preservation gives more variable results (Table 4). The three

lyophilisation protocols that more affect growth are PML2

(60 % of the isolates being affected), MTL2 and PTL2 (47 %).

The cryopreservation protocols that more affect growth are

MGC2 (53 % of isolates being affected), MC1, MTC2 and PGC2

(47 %).

Growth rate stimulation or inhibition, following lyophilisa-

tion or cryopreservation, is not species-dependent. Actually

several isolates belonging to the same species could react in

a different way (Table 5).

Enzymatic activity – The analysis of Lac, MiP and MnP activ-

ities as the frequency of significant differences from control

values are summarized in Table 4. Even if lyophilisation

causes the greatest effect on enzymatic activities, with the ex-

ception of MiP activity, the effects of stimulation or inhibition

induced by preservation protocols do not follow a general

trend. Lac activity was the most influenced by all preservation

methods leading to either increases or decreases of activity.

Table 5 – Physiological analysis of 15 white-rot isolates after one month of preservation under six cryopreservation andeight lyophilisation protocols

n: activity not detected in control and treated samples; [Y: significant increase ([) o reduction (Y) of the tested parameter; ¼: no significant

differences from the control value, (Mann–Whitney test p� 0.05).

a G: growth rate; Lac: laccase activity; MiP: Peroxidase Mn-independent activity; MnP: Peroxidase Mn-dependent activity.

b y: death after preservation.

Fig 1 – PCoA arising from the morphological and physio-

logical data for 15 controls and 15 treated isolates preserved

by six protocols of cryopreservation at L80 �C and by eight

protocols of lyophilisation.

Vitality and genetic fidelity of white-rot fungi following preservation 1035

Nevertheless, the enzymatic activity analysis demonstrated

that none of the activities evaluated were lost after preserva-

tion. After lyophilisation, the percentage of isolates that dis-

played significant differences of Lac activity ranged between

26 (MTL2) and 72 % (PML3). For MML2, MML3 and PML3 signif-

icant differences occurred in 55 %, 63 % and 72 %, respectively.

After cryopreservation, the percentage of isolates that dis-

played significant differences of Lac activity ranged between

20 (MGC2) and 53 % (PGC2). MiP and MnP activities were also

affected by all preservation methods with uneven results.

The PML2 and MC1 preservation protocols have had the

most important effect on MiP activity; 30 % and 33 % of the iso-

lates being affected, respectively. Regarding MnP, MTL2 and

PTL2 (13 % of isolates affected for both methods), and MTC2

(20 % of isolates affected) have had the biggest effect on MnP

activity. The effect of preservation (stimulation or inhibition)

on the enzymatic activities tested is not correlated to species,

as demonstrated by the fact that isolates belonging to the

same species could react in a different way (Table 5).

PCoA analysis – The PCoA analysis, obtained on the basis of

the complete set of data [vitality, growth end point (6 repli-

cates), morphology and enzymatic activity (5 replicates for

each tested enzyme)], shows that all cryopreservation methods

cluster with the control more than observed for the lyophilisa-

tion ones. The two lyophilisation methods MTL2 and PTL2 are

close to the control while all the others are scattered (Fig 1).

Evaluation of genetic fidelity – The analysis of the ribosomal

DNA internal transcribed spacers sequences (ITS) of the two

G. adspersum isolates (MUT 3426 and MUT 3427), that displayed

morphological alterations after lyophilisation, revealed that

no contamination occurs during the lyophilisation process

(data not shown).

For each isolate (control or treated) a multilocus DNA fin-

gerprint was generated with the four AFLP primer pairs. The

DNA fragments were scored between 60 and 500 base pairs

in length and 150 (S. paradoxa MUT 3390) up to 298 fragments

were scored (Trametes gibbosa MUT 3364). The total number of

DNA fragments obtained for each control isolate and the dif-

ferences between control and treated isolates fingerprint pro-

files are indicated in Table 6.

Thetechnicalerror raterangesfromaminimumvalueof3.9%

to a maximum of 5.4 %. After cryopreservation by protocol vari-

ant PC1, the 15 tested isolates displayed differences in fingerprint

profiles inside the range of the technical error. Moreover, for 13

out of 15 isolates, differences were lower than 3.9 %. Regarding

the lyophilisation variant MTL2, 14 isolates displayed fingerprint

differences inside the technical error range, and for 13 out of 15

isolates differences were lower then 3.9 %. After lyophilisation

using the protocol PTL2, both strains of G. adspersum MUT 3426

and MUT 3427 displayed fingerprint differences outside the tech-

nical error rate and only 7 out of 15 isolates displayed fingerprint

differences lower then the 3.9 % (Table 6).

Discussion

The first aim of this study was to set up different protocols of

lyophilisation and cryopreservation at �80 �C on different

species of WRF and, subsequently, to check isolates vitality

and the maintaining of some morpho-physiological features

(macro and microscopic morphology, growth, enzymatic

activities). The second aim was to select one protocol of

cryopreservation at�80 �C and at least one protocol of lyophi-

lisation, and to investigate the eventual presence of genetic

variation by means of AFLP analysis.

Regarding vitality, our results have demonstrated that

cryopreservation at �80 �C could be useful for mid-term pres-

ervation of some WRF, as all fungal isolates tested in this work

were successfully preserved by all cryopreservation methods

after one month of storage at �80 �C. Our study did not reveal

any influence of growth media composition, growth modali-

ties, cryoprotectant, time and type of perfusion, samples prep-

aration, and thawing temperatures, on vitality.

The WRF species used in this work, with the only exception

of Schizophyllum commune, have never been reported in litera-

ture as suitable for lyophilisation. The variant protocols MTL2

and PTL2 gave the best results among all lyophilisation proto-

cols tested, both in terms of vitality with percentages for WRF

up to 100 %, and considering the PCoA analysis. In the method

L2, isolates are grown on liquid culture media containing lyo-

protectant (trehalose for PTL2 and MTL2) in addition to the

double perfusion. This could have improved the trehalose ab-

sorption, providing to mycelia not only an energy source, but

also a massive accumulation of intracellular trehalose avail-

able for other functions. It is well known that in fungal cells

trehalose plays a lot of very important functions, linked with

the biology of growth and development, in normal and

stressed conditions (Arguellas 1997; Doehlemann et al. 2006;

d’Enfert et al. 1999; Fillinger et al. 2001; Jepsen & Jensen 2004;

Managbanag & Torzilli 2002; Ocon et al. 2006; Tereshina

2005). In addition, trehalose has a stabilizing effect on mem-

branes during freezing and drying and its ability to stabilize

proteins during heat shock has been demonstrated (Tereshina

2005). These findings are in agreement with the data of Croan

(2000), who successfully lyophilised tropical wood-inhabiting

basidiomycetes, demonstrating that the survival rates in-

creased considerably when isolates were allowed to growth

in medium containing trehalose, and when trehalose was

added in the lyophilisation suspending medium.

Table 6 – Differences between AFLP fingerprints profilesof control and treated white-rot isolates

Species MUTN�

Number of DNAfragments scored

in controlsamples

DNA fingerprintdifferencesa (%)

PC1b MTL2c PTL2d

C. gallica 3379 178 1.7 0.6 1.7

C. gallica 3380 188 2.1 1.6 3.7

C. gallica 3382 187 0.5 0.0 0.0

D. confragosa var.

confragosa

3483 159 3.5 2.0 1.0

G. adspersum 3426 269 3.7 26.0 23.8

G. adspersum 3427 209 1.0 0.5 26.1

L. betulina 3368 155 3.2 1.3 0.0

P. crispa 3394 195 0.5 1.5 4.1

P. crispa 3496 174 4.7 4.7 4.1

S. commune 3392 247 0.0 0.0 1.2

S. paradoxa 3389 150 4.0 1.3 5.4

S. paradoxa 3390 235 2.1 1.3 4.3

T. gibbosa 3364 298 1.3 0.3 0.3

T. pubescens 2400 171 2.9 2.9 4.1

T. versicolor 3374 201 1.0 3.0 4.5

MUT¼Mycotheca Universitatis Taurinenesis.

a DNA fingerprint differences, differences outside the range of the

technical error (3.9–5.4 %) are marked in bold characters.

b Cryopreservation protocol PC1.

c Lyophilisation protocol MTL2.

d Lyophilisation protocol PTL2.

1036 S. Voyron et al.

Our morpho-physiological analyses showed that, following

cryopreservation at �80 �C or lyophilisation, most WRF iso-

lates did not reveal major changes in their morphological

and physiological features. Only two Ganoderma adspersum iso-

lates (MUT 3426 and MUT 3427) demonstrated changes in mor-

phological features after lyophilisation, as a strong hyphae

vacuolization, as well as the irregular growth observed in

the isolate MUT 3426. Vacuoles are known or suspected to

play several crucial roles in the physiology of vegetative

growth (Weber 2002). Only some of these roles are related to

the activity of lytic intravacuolar enzymes. Indeed, vacuoles

are involved in apoptosis process: a high vacuolization is asso-

ciated with programmed cell death (Lu 2006) and in vegetative

incompatibility (Glass et al. 2000). They are also the destina-

tion of endocytic vesicles originating by inward budding of

the plasma membrane, thereby providing mechanisms of

‘‘face lifting’’ of the plasma membrane and a mean of commu-

nication with the extracellular environment (Weber 2002).

Further vacuoles play a key role in homeostasis; actually

they store organic and inorganic nutrients, may detoxify the

cytoplasm by sequestration of toxic substances, and are in-

volved in the maintenance of a balanced chemical composi-

tion of the cytoplasm in face of fluctuating external

conditions (Weber 2002). The strong vacuolization observed

in revived isolates of G. adspersum (MUT 3426 and MUT 3427)

after lyophilisation, could be interpreted as sign of cellular

suffering. It could be the cellular answer to a malfunction of

some metabolic pathway, which lead to an unbalanced ho-

meostasis, and may mirror some genetic mutation.

A genetic mutation could be also suspected for the change

of the aerial mycelium observed in the isolate G. adspersum

MUT 3426. Many studies have established a link between ex-

pression of hydrophobins and the ability of the fungus to

grow aerial structure (Linder et al. 2005). Hydrophobins have

been shown to occur in many filamentous fungi (Linder et al.

2005), where they can also take part in a broad spectrum of

other biological function during fungal morphogenesis, path-

ogenesis and symbiosis; their expression is under the control

of complex factors (Linder et al. 2005; Ma et al. 2007; Whiteford

& Spanu 2002). On these bases we can suppose that the mor-

phological variations that involve aerial mycelium growth of

G. adspersum MUT 3426, following lyophilisation by MTL2

and PTL2, could be due to a modification in hydrophobins reg-

ulation, or could reflect a genetic mutation of some of the

genes of the hydrophobins family.

From a more general point of view, our results emphasize

that the application of a preservation protocol, or one of its

variants, does not always give the same results on the totality

of tested isolates, although they belonged to the same species.

These findings confirm that the response to preservation pro-

tocol could not be only genus-dependent or species-depen-

dent but also isolate-dependent (Smith & Onions 1983, 1994;

Ryan et al. 2000).

On the basis of the PCoA results, of the longevity, of the fa-

cilities and on the labour involved, one protocol of cryopreser-

vation at �80 �C (PC1), and two of lyophilisation (MTL2 and

PTL2), were selected among all the protocols tested. The pro-

tocol PC1 was chosen because it is less expensive than MC1,

and also because it is a ‘‘save space’’ protocol, as three copies

of each isolate can be preserved in one cryotube, while, with

all other variants of the cryopreservation protocol C2, each

cryotube contains only one copy. The protocols MTL2 and

PTL2, that differed for the mycological media used to grow iso-

lates prior lyophilisation, were chosen owing their close prox-

imity to controls in the PCoA plot and in consequence of the

lack of data in literature about basidiomycetes preservation

by lyophilisation.

The possible presence of modifications at genetic level was

checked by means of AFLP fingerprinting (Vos et al. 1995). AFLP

is one of the most robust fingerprinting methods among ge-

netic marker techniques that have been developed for geno-

typic characterization. AFLP analysis have been also used to

assess the genetic fidelity of some plant species after different

conservation techniques such as cold storage, slow growth,

micropropagation, cryopreservation and cryopreservation by

vitrification (Hao & Deng 2005; Liu et al. 2003; Turner et al.

2001; Wilkinson et al. 2003) and to evaluate the quality of

boar sperm following cryopreservation (Thurston et al. 2002).

The comparison between AFLP fingerprints of the 15 con-

trol and the 15 cryopreserved isolates reveals that no differ-

ences outside the range of the technical error were

observed. Moreover, 13 out of 15 isolates displayed fingerprint

differences lower than the lower technical error value. These

results, coupled with those obtained by the morphological and

physiological analysis, provide evidence that the cryopreser-

vation protocol PC1 at �80 �C is useful for mid-term preserva-

tion of the species studied in this paper. Concerning

lyophilisation, no effect on genetic fidelity was detected for

14 out of 15 treated isolates with the lyophilisation protocol

MTL2 and for 13 out of 15 isolates with protocol PTL2. Only

the two isolates of G. adspersum (MUT 3426 with MTL2 and

Vitality and genetic fidelity of white-rot fungi following preservation 1037

PTL2 and MUT 3427 with PTL2) displayed fingerprint differ-

ences between control and treated isolates outside the range

of the technical error. Although two isolates fail to retain their

genetic characteristics following lyophilisation, the most im-

portant result is that the majority of WRF were able to retain

their genetic features after preservation. These findings, cou-

pled with the results obtained by the morphological and phys-

iological analysis, give us a sufficient degree of confidence in

the reliability of the two lyophilisation protocols selected.

Finally, for all isolates preserved by the three protocols se-

lected in this study (PC1, MTL2 and PTL2), we didn’t report any

loss of vitality after 18 m of preservation (data not shown).

These results, even if obtained on a small number of WRF spe-

cies, are encouraging. Further investigations on a large num-

ber of WRF species, and for long periods of conservation,

will allow to evaluate the suitability of the protocols optimized

in this study for long-term (lyophilisation) or mid-term (cryo-

preservation at �80 �C) preservation of WRF.

Acknowledgments

The authors wish to thank Stephanie Huret of the Mycotheque

of the Universite catholique de Louvain for her much appreci-

ated technical assistance, and Simone Priante of the Depart-

ment of Chemistry IFM of the University of Turin for the

DSC analysis.

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