International Journal of Food Microbiology 137 (2010) 204–213
Contents lists available at ScienceDirect
International Journal of Food Microbiology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Microsatellite loci to recognize species for the cheese starter and contaminating
strains associated with cheese manufacturing
Frédéric Giraud a, Tatiana Giraud b,c, Gabriela Aguileta b,c, Elisabeth Fournier d, Robert Samson e,
Corine Cruaud f, Sandrine Lacoste a, Jeanne Ropars a, Aurélien Tellier g, Joëlle Dupont a,⁎
a
Muséum National d'Histoire Naturelle, Département Systématique et Evolution, UMR OSEB 7205, CP 39, 57 rue Cuvier, 75231 Paris Cedex 05, France
ESE, Bâtiment 362, Université Paris-Sud, 91405 Orsay cedex, France
CNRS, 91405 France
d
UMR BGPI, TA A 54/K, Campus International de Baillarguet, 34398 Montpellier Cedex 5, France
e
CBS, P.O.Box 85167, 3508 AD Utrecht, The Netherlands
f
Genoscope, Centre National de Séquençage, 2, rue Gaston Crémieux, CP5706, 91057 Evry Cedex, France
g
Section of Evolutionary Biology, LMU BioCenter, Grosshaderner Str. 2, 82152 Planegg-Martinsried, Germany
b
c
a r t i c l e
i n f o
Article history:
Received 3 July 2009
Received in revised form 5 November 2009
Accepted 20 November 2009
Keywords:
Contaminant
Domestication
PC4 marker
Multigenic phylogeny
Penicillium
Starter cultures
a b s t r a c t
We report the development of 17 microsatellite markers in the cheese fungi Penicillium camemberti and
P. roqueforti, using an enrichment protocol. Polymorphism and cross-amplification were explored using 23
isolates of P. camemberti, 26 isolates of P. roqueforti, and 2 isolates of each of the P. chrysogenum and P. nalgiovense
species, used to produce meat fermented products. The markers appeared useful for differentiating species, both
using their amplification sizes and the sequences of their flanking regions. The microsatellite locus PC4 was
particularly suitable for distinguishing contaminant species closely related to P. camemberti and for clarifying the
phylogenetic relationship of this species with its supposed ancestral form, P. commune. We analyzed 22 isolates
from different culture collections assigned to the morphospecies P. commune, most of them occurring as food
spoilers, mainly from the cheese environment. None of them exhibited identical sequences with the ex-type
isolate of the species P. commune. They were instead distributed into two other distinct lineages, corresponding
to the old species P. fuscoglaucum and P. biforme, previously synonymised respectively with P. commune and
P. camemberti. The ex-type isolate of P. commune was strictly identical to P. camemberti at all the loci examined.
P. caseifulvum, a non toxinogenic species described as a new candidate for cheese fermentation, also exhibited
sequences identical to P. camemberti. The microsatellite locus PC4 may therefore be considered as a useful
candidate for the barcode of these economically important species.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
The genus Penicillium (Ascomycota) is well known for its importance in cheese industry. In addition to the emblematic species
P. camemberti and P. roqueforti, used as starters for the production of
many cheeses and essential to their taste, some other species appear
important, as major contaminants in cheese manufactures. Among
them are P. commune, P. solitum, P. palitans and P. crustosum (Lund
et al., 1995, 2003; Kure and Skaar, 2000; Kure et al., 2001, 2003, 2004;
Samson and Frisvad, 2004; Dupont et al., unpublished data). The
problem for manufacturers is the close genetic relationships among
starter strains and contaminants. P. camemberti for instance, used
in the production of French soft cheeses, e.g., Camembert, Brie or
⁎ Corresponding author.
E-mail address: [email protected] (J. Dupont).
URL: http://www.genoscope.fr (C. Cruaud).
0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2009.11.014
Neuchatel, is regarded as a domesticated species derived from the
contaminant species P. commune (Pitt et al., 1986). P. roqueforti, used
for the fermentation of the blue Roquefort cheese, appears as a
contaminant in hard cheeses (like Emmental and Parmesan) factories
(Dupont et al., unpublished data). The limits between the contaminant
or the biotechnological status of isolates are thus very tenuous and an
accurate delimitation of species is essential, both for monitoring the
production process and for the identification of spoilage fungi.
Fungal species have traditionally been diagnosed on the basis of
morphology alone. The use of multiple phenotypic characters, including growth on different media and at different temperatures, or
pigment production, has been very useful for species delimitation in
Penicillium (Pitt, 1979). However, fungal taxonomists now routinely
use the concordance of different gene genealogies (GCPSR: Genealogical Concordance Phylogenetic Species Recognition criterion) to
delimit species because it appears congruent with, and more finely
discriminating than, morphology and interfertility species recognition
criteria (e.g., Taylor et al., 2000; Koufopanou et al., 2001; Dettman
205
F. Giraud et al. / International Journal of Food Microbiology 137 (2010) 204–213
et al., 2003; Pringle et al., 2005; Le Gac et al., 2007a,b; Giraud et al.,
2008a). After species delimitation using the GCPSR criterion, DNA
barcode markers can be chosen for rapid species identification (see
All Fungi Barcoding: http://www.allfungi.com/).
In mycology, ITS rDNA has been widely used for species recognition and barcoding but is not diverse enough to delimit species of
the cheese environment (Boysen et al., 1996; Pedersen et al., 1997;
Skouboe et al., 1999). Geiser et al. (2000) have searched for more
variable markers to discriminate closely related species of Penicillium
and Aspergillus and have shown that β-tubulin exhibits an appropriate level of divergence between species in these genera. In a broad
study of terverticillate Penicillia, Samson et al. (2004) showed using
the β-tubulin that species contaminating cheese, P. palitans, P. solitum
and P. crustosum, each formed a separate clade. In contrast, P. commune
did not appear monophyletic: the ex-type strain of P. commune was
placed within the P. camemberti clade while other P. commune isolates
formed a separate clade. PCR typing methods (RAPD, AFLP and PCR
fingerprinting using M13 primer) have been used to describe the
distribution of isolates of the contaminant species P. commune and
P. palitans in several cheese factories (Kure et al., 2002, 2003; Lund
et al., 2003). RAPD did not discriminate cheese starter cultures
(Dupont et al., 1999; Geisen et al., 2001). AFLP showed a better
discriminatory power than M13 fingerprinting and RAPD, revealing
up to 55 AFLP groups among 321 P. commune isolates identified based
on phenotypic characters (Lund et al., 2003). AFLPs are however timeconsuming, not always repeatable between laboratories, and the
polymorphic bands may not behave as Mendelian markers (Dutech
et al., 2007). Other markers were therefore needed. Microsatellites
have been shown to be powerful for strain-specific identification in
fungi (Marinangeli et al., 2004; Mathimaran et al., 2008), and their
flanking regions have been used to recognize fungal species in several
complexes of pathogenic species (Fisher et al., 2000; Pringle et al.,
2005; Matute et al., 2006; Giraud et al., 2008c).
In this study, 23 microsatellite loci were therefore developed (11
for P. roqueforti and 12 for P. camemberti). Sequences flanking six
microsatellite loci were used together with four protein coding regions
to recognize species for the cheese starter and contaminating strains
associated with cheese manufacturing. The utility of microsatellites
was also examined for distinguishing among Penicillium isolates used
as starter cultures in food production.
2. Materials and methods
Table 1
Isolates from culture collections used in this study, with revised molecular identification.
Species
Isolate number
Substrate
Origin
P. biforme
CBS 297.48 T
LCP 75.2621
LCP 08.5496
LCP 08.5497
LCP 08.5498
LCP 08.5499
LCP 08.5500
LCP 08.5501
LCP 08.5502
CBS 279.67
CBS 299.48 T
= LCP 66.584 T
= MUCL 29790 T
= NRRL 877 T
LCP 66.1920
French cheese
Unknown
Cheese factory
Cheese factory
Cheese factory
Cheese factory
Cheese factory
Cheese factory
Cheese factory
Roquefort cheese
French Camembert
cheese
Connecticut, USA
Unknown
France
France
France
France
France
France
France
Netherlands
Connecticut, USA
French St Marcellin
cheese
French St Marcellin
cheese
Dried sausage
Caen, France
P. camemberti
LCP 75.3054
LCP 98.4258
CMPG 30
CMPG 470
CMPG 602
CBS 112078
CBS 190.67
(ex P. rogeri)
(ex P. candidum)
(ex P. caseicola)
P. caseifulvum
P. commune
2.3. Isolation of microsatellite loci
Three microsatellite enriched-libraries were built according to
Giraud et al. (2002) using biotin-labelled microsatellite oligoprobes
and streptavidin-coated magnetic beads. Total genomic DNA was
extracted from one isolate of P. roqueforti, and one isolate of
MUCL 34882 T
= NRRL 890 T
CBS 216.30 NT
P. commune
(ex P. lanosogriseum)
P. fuscoglaucum
LCP 91.2798 T
= NRRL 892 T
= MUCL 28651 T
= CBS 261.29 T
LCP 50.218
LCP 52.797
LCP 79.3239
CMPG 74
LCP 06.5327
LCP 07.5471
2.1. Fungal isolates
Two sets of fungal isolates were used in this study: isolates from
culture collections (Table 1), including strains of species that have
been synonymised with P. commune and P. camemberti, were used for
taxonomic purposes; biotechnological isolates provided by producers
(Table 2) were used to assess the possibility of cross-amplifications
and to investigate amplification size variability at microsatellite loci.
Isolates numbered LCP 08.5496 to LCP 08.5502 are under confidential
safe deposit and are therefore not available. Sequences of the ex-type
strains of P. camemberti, P. commune and P. fuscoglaucum were checked
using isolates preserved in different culture collections (CBS, LCP, and
MUCL), in particular in NRRL, the original depository collection.
Isolates were cultivated on Malt Extract Agar (MEA) and Czapeck
Yeast Agar (CYA) and incubated at 10, 15, 25, 30 and 37 °C during 7 days
for colony diameter measurements and morphological observations.
CBS 112325
CBS 123.08 T
MUCL 29157 T
CBS 303.48 T
CBS 101134 T
CBS 108956
CBS 112324
CBS 112881
LCP 07.5472
CBS 112079
CBS 111835
P. palitans
CBS
CBS
CBS
CBS
101031
115507
112204
491.84
LCP 61.1628
LCP 04.4862
P. crustosum
LCP 75.3045
CBS 101025
CBS 471.84
CBS 181.89
CBS 313.48
French blue Cheese
Goat cheese
Inoculum for dried
sausage
Cheese
contaminant
Dutch camembert
cheese
Unknown
Camembert cheese
Unknown
Camembert cheese
Danablue cheese
Cheese
Unknown
Food waste
(compost)
Cheese
France
Haute-Loire,
France
France
Grenoble, France
Marseille, France
Switzerland
Netherlands
Unknown
France
Unknown
France
Denmark
Denmark
Denmark
Germany
Connecticut, USA
Leaf litter
Netherlands
Unknown
Unknown
Rubber
Washing water,
textile industry
Wood
Walnut
Yogurt
Refrigerated
equipment
Refrigerated
equipment
Feta cheese
Mummified
bee larva
Cocoa
Unknown
Unknown
Mouldy liver paste
Vietnam
Unknown
Netherlands cheese
Mixed cereal and
honey
Animal feed
Cheese
Thymus sp.
Soil with Agaricus
bisporus
Oryza sativa
Unknown
France
France
France
France
Denmark
USA
Japan
Japan
Russia
Holbeck ,
Denmark
France
Grenoble, France
Azores, Portugal
Denmark
Denmark
Fiji
LCP : Laboratoire Cryptogamie Paris, CBS : Centraalbureau voor Schimmelcultures,
MUCL : Mycothèque Université Catholique Louvain, NRRL : Northern Regional Research
Laboratory, CMPG : Collection Mycologie Pharmacie Grenoble, T : type , NT neotype.
206
F. Giraud et al. / International Journal of Food Microbiology 137 (2010) 204–213
Table 2
List of biotechnological isolates used to evaluate polymorphism of microsatellite loci. Alleles are numbered from 0 to 5, 0 being a null allele (lacking amplification product), 1–5
numbered according to their decreasing length. Cross amplification are mentioned by +.
Species
Isolate number
Producers
P. camemberti
B003306
TE0EL
P. camemberti
B000952
DREWES
P. camemberti
B000911
SKW
P. camemberti
B001108
SKW
P. camemberti
B001109
SKW
P. camemberti
B001110
SKW
P. camemberti
B001112
SKW
P. camemberti
B001113
SKW
P. camemberti
B001116
SKW
P. camemberti
B001152
SKW
P. camemberti
B000956
SOCHAL
P. camemberti
B000957
SOCHAL
P. camemberti
B000936
TE0EL
P. camemberti
B000937
TE0EL
P. camemberti
B000940
TE0EL
P. camemberti
B000942
TE0EL
P. camemberti
B000945
TE0EL
P. camemberti
B000948
TE0EL
P. camemberti
B000949
TE0EL
P. camemberti
B001378
TE0EL
P. camemberti
B002770
VISBY
P. camemberti
B002771
VISBY
P. camemberti
B002772
VISBY
P. chrysogenum B000766
SKW
P. chrysogenum B000767
SKW
P. nalgiovense
B000953
SKW
P. nalgiovense
B003307
DREWES
P. roqueforti
B001349
TE0EL
P. roqueforti
B001350
CSL
P. roqueforti
B001351
CSL
P. roqueforti
B001352
CSL
P. roqueforti
B001353
CSL
P. roqueforti
B001228
SOREDAB
P. roqueforti
B001524
SOREDAB
P. roqueforti
B001460
SKW
P. roqueforti
B001371
STANDA LRL
P. roqueforti
B001372
STANDA LRL
P. roqueforti
B001373
STANDA LRL
P. roqueforti
B001374
STANDA LRL
P. roqueforti
B001376
STANDA LRL
P. roqueforti
B001355
SWING
P. roqueforti
B001356
SWING
P. roqueforti
B000935
TE0EL
P. roqueforti
B000944
TE0EL
P. roqueforti
B000946
TE0EL
P. roqueforti
B001340
VISBY
P. roqueforti
B001343
VISBY
P. roqueforti
B001344
VISBY
P. roqueforti
B001345
VISBY
P. roqueforti
B001346
VISBY
P. roqueforti
B001347
VISBY
P. roqueforti
B001348
VISBY
P. roqueforti
B001377
VISBY
Cross amplification/Total number of alleles
PC1
PC2
PC3
PC4
PC5
PC6
PC7
PC8
PC9
PC10
PC11
PC12
PC13
PR4b
PR5
PR6
PR7
1
1
1
1
nd
nd
1
1
1
nd
1
1
1
1
1
1
1
1
1
1
1
1
1
0
nd
0
nd
2
2
2
nd
2
2
2
2
2
nd
nd
nd
2
nd
nd
nd
2
2
nd
nd
nd
nd
nd
2
nd
nd
+/3
1
1
1
1
1
1
1
1
1
nd
1
1
1
1
1
1
1
1
1
1
1
1
1
nd
nd
2
nd
3
3
3
nd
3
3
3
3
3
nd
nd
nd
3
nd
nd
nd
3
3
nd
nd
nd
nd
nd
3
nd
nd
+/4
1
1
1
1
1
1
nd
nd
1
nd
1
1
1
1
1
1
1
1
1
1
1
1
1
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
1
1
1
1
1
1
1
1
1
1
nd
nd
nd
1
nd
1
1
1
1
1
nd
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
nd
nd
nd
nd
nd
nd
nd
1
1
nd
1
1
1
nd
nd
nd
nd
nd
nd
nd
1
1
1
0
nd
0
nd
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
nd
1
1
1
1
1
1
1
nd
nd
1
1
1
nd
1
nd
1
1
nd
nd
nd
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
1
1
1
1
nd
1
1
nd
nd
nd
1
nd
nd
nd
1
1
1
1
1
1
1
nd
nd
0
nd
0
nd
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
1
1
1
1
1
nd
nd
nd
1
nd
1
1
nd
1
1
1
1
1
nd
1
1
1
1
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+/3
2
2
2
2
2
2
2
nd
nd
nd
2
2
2
2
2
2
2
2
2
2
nd
2
2
0
0
0
2
nd
nd
nd
nd
1
1
1
1
1
1
nd
1
1
1
nd
1
1
1
1
1
1
1
1
1
1
1
+/3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
3
3
3
3
nd
nd
nd
nd
2
2
2
2
2
2
nd
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
+/3
1
1
1
1
1
1
1
1
1
nd
1
1
nd
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
1
1
1
1
1
1
1
1
1
nd
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
nd
nd
nd
nd
3
3
3
3
3
3
nd
3
3
3
3
3
3
3
3
3
3
nd
nd
nd
3
3
+/3
nd
nd
nd
nd
nd
3?
nd
4?
3?
nd
3?
3?
4?
nd
3?
3?
nd
4?
4?
nd
4?
4?
3?
5
nd
5
nd
1
1
1
nd
1
1
1
1
1
nd
nd
nd
2
2
nd
nd
1
1
nd
nd
nd
nd
nd
2
nd
nd
+/5
2?
1?
1?
2?
nd
nd
nd
2?
nd
nd
1?
nd
1?
nd
1?
nd
2?
2?
1?
nd
nd
nd
1?
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
+/3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
nd
nd
nd
1
1
1
1
1
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
+/3
nd: no data, ? : doubt on the size.
P. camemberti. Three enriched libraries were constructed using the
P. roqueforti isolate (called hereafter PR1, PR2 and PR3 libraries), and
one library using the P. camemberti isolate (called hereafter PC
library). The PR1 and PC libraries were built using the oligoprobes
(TG)10 and (AAG)10, whereas the PR2 library was built using the
oligoprobes (TG)10 and (TC)10, and the PR3 library was built using the
probes (CAC)10 and (CCT)10. In the PR1 enriched library, 900 clones
were screened and 30 gave a positive response (3.3 %). In the PR2
enriched library, 400 clones were screened and 79 gave a positive
response (20 %). In the PR3 enriched library, 200 clones were screened
and 2 gave a positive response (1 %). In the PC enriched library, 1000
clones were screened and 54 gave a positive response (5.4 %). A total
of 51 clones were sequenced, 5 (10 %) of which were found to be
redundant and contaminant from a previous enrichment (Giraud
et al., 2002).
PCR primers were designed for 23 loci (9 from the PR1 library, 2
from the PR2 library, and 12 from the PC library), using the computer
program OLIGOTM (Macintosh version 4.0, National Bioscience). Each
locus was screened for amplification in species of starter cultures and
for variation among the strains using a panel of 53 biotechnological
isolates (Table 2). These isolates included 23 isolates of P. camemberti,
2 isolates of P. chrysogenum, 2 isolates of P. nalgiovense and 26 isolates
of P. roqueforti. PCR amplifications were performed using a Biometra
thermal cycler, with 35 cycles of 94 °C for 30 s, 50 °C for 30 s, and
72 °C for 30 s. Each reaction (10 µl) contained 1 µl of 10× reaction
buffer (50 mM KCl, 0.1% Triton X-100, 10 mM Tris–HCl, pH 9.0),
F. Giraud et al. / International Journal of Food Microbiology 137 (2010) 204–213
75 µM of dCTP, dGTP, dTTP, 6 µM of dATP, 0.02 µl of 33P dATP, 0.2 µg/µl
BSA, 1.5 mM MgCl2, 2.5 pmol of each primer, 0.25 U of Taq DNA
polymerase (Promega), and approximately 10 ng of sample DNA. PCR
products were analysed in 6% polyacrylamide gels and visualized by
autoradiography.
2.2. DNA extractions, PCR amplifications and sequencing
Genomic DNA was extracted from fresh mycelium grown from
isolates listed in Table 1 on Malt Agar for 5 days using a CTAB
micropreparation method (Rogers and Blendich, 1985). Most of the
CBS isolates were kindly provided as DNAs by R. Samson. PCR was
performed in 50 µl reactions, using 25 µl of template DNAs, 1.25 U of
AmpliTaq DNA polymerase (Roche Molecular Systems, Inc., Branchburg, NJ, USA), 5 µl of 10× Taq DNA Polymerase buffer, 5 µl of 50 %
glycerol, 2 µl of 5 mM dNTPs (Eurogentec, Seraing, Belgium), 2 µl of
each 10 µM primer and 50–100 ng template DNA.
The oligonucleotide primer sets used in this study were : Bt2a and
Bt2b (Glass and Donaldson, 1995) to amplify a part of the 5′ end of
the ß-tubulin (TUB) gene; CF4/CF1D and CF4/CFM to amplify a portion
of the calmodulin (CAL) gene (Peterson, 2004); the universal primer
set EF6 and EF1D to amplify a part of the 5′ end of the translation
elongation factor 1-alpha (EF-1α) (Peterson, 2004); Pen F1/Pen R1 or
PenF1/Asp R1 to amplify the COI fragment (Seifert et al., 2007); PC4F
and PC4R to amplify the microsatellite locus PC4 isolated in this study
(Table 3).
Amplifications were performed on a Perkin Elmer Cetus thermal
cycler model 2400 with 30 cycles of 30 s at 94°, 30 s at 55° and 40 s at
72° for TUB, 42 cycles of 30 s at 94°, 30 s at 51° and 90 s at 72° for CAL
Table 3
Information on the 17 microsatellite loci used in this study.
Microsatellite
locus a
Repeat
motif b
Primers sequences (5′-3′)
Annealing
T (°C)
Size
(bp)
PR4 Bis
(GA)12
53
139
PR5
(TG)8
52
128
PR6
(GA)10
53
147
PR7
(TG)17
59
260
PC1
(TC)6 + (CA)7
52
193
PC2
(CA)9
60
225
PC3
(TG)6
58
207
PC4
(AG)11
54
198
PC5
(AG)12
51
232
PC6
(AG)14
52
183
PC7
(AG)33
55
160
PC8
(TC)n
51
206
PC9
(TG)9
53
174
PC10
(TG)n
56
300
PC11
(AC)8
51
168
PC12
T10A9
51
155
PC13
(TC)13
F : cag gcg tta gtg cgt tca aa
R : acc aac gat acg caa ccg at
F : ccc tgc ttg ttg gat tgt cg
R : taa ctt tga gag ggt cgc ct
F : ggc cgc att gta agt cat tc
R : tta gga tgg ttc tcg ggt ca
F : caa gcc agc tca gga aac ga
R : cgt gtt gga gtt cga gcc ga
F : tcc cag atc aac gcc caa ca
R : gag tcg ggg gtg atg atg cg
F : gga agt tca gct cgt tcc ag
R : ggg cgc att atg atg ttt tt
F : ccg act cgg cct ttt tgg
R : caa gca gag cct cgt att cc
F : caa gct ggc cga taa cct ga
R : cca tcc gct tga ttt ctc ct
F : gga tga agt ctg tgg gaa gg
R : cct tcc cac aga ctt cat cc
F : tgt att gcc tga tgc cat tc
R : gca cac aag gca gaa ata tgc
F : cag cca gtc gac cgt ata cg
R : cta agt gct cgg cca acg at
F : ggg cag cag tag agg gat ag
R : caa cat cac atg ccg aat ga
F : ggc caa aag cat gtt gaa gt
R : gta ggc tgc ata tcg ttt cg
F : taa ccc gta aac ccg taa cc
R : caa caa act cgc acg agg gg
F : agc caa ctg cat gtg ata cac
R : ctc caa tca cga gca tgt atg a
F : agt ggg ctt cag tct cct tg
R : aat act gcc ctc tca cgc aa
F : cca tcc gct tga ttt ctc ct
R : cca att cct gga tat caa cat
51
154
a
Loci named PR and PC were cloned respectively in the PR and PC libraries.
The sign “+” indicates that two microsatellite motifs were separated by several
base pairs in the clone.
b
207
and EF-1α, 35 cycles of 30 s at 94°, 30 s at 50 °C and 30 s at 72 °C
for PC4 and 35 cycles of 30 s at 94°, 30 s at 56 °C and 30 s at 72 °C for
COI, followed by a final 10 min extension step at 72°. PCR products
were purified and sequenced by GenoScreen (Lille, France) or by the
Genoscope (Évry, France), in both directions to confirm the accuracy
of each sequence. Sequences have been deposited in the GenBank
database under accession numbers FJ930933 to FJ930986 for the
TUB sequences, FJ930987 to FJ931040 for the EF-1α sequences and
EU003130 to EU003169 for PC4 sequences.
2.4. Phylogenetic analysis
Sequences were aligned with ClustalW using MEGA software
(Kumar et al., 2004). Alignments were edited manually. Kimura twoparameter genetic distances (d = number of nucleotide differences
per site between two sequences, Kimura, 1980) were calculated using
MEGA. Sites with indels were removed from multiple sequence alignments using Genedoc (Nicholas et al., 1997). The program PHYML
(Guindon and Gascuel, 2003) was used to build the phylogenetic trees
under a maximum likelihood framework. We used model test (Posada
and Crandall, 1998) to choose the best nucleotide substitution model,
namely, HKY85 allowing for among-site rate variation by using the
discrete gamma model with four rate classes. Also, we estimated
and optimized the proportion of invariable sites and the transition/
transversion rate ratio. Support for the branches was determined
from 1000 bootstrap replicates using the majority rule criterium in
Consense in the PHYLIP package (Felsenstein, 1989).
For the microsatellite locus PC4, the number of repeats of the motif
was species specific and we coded the repeat variation in the microsatellite region according to the ID Code (Barriel, 1994). To take
advantage of the indels present in the data, maximum parsimony was
used to infer phylogeny from the PC4 locus using PAUP.
Penicillium crustosum, a member of the series Solita, a close relative
of series Camemberti, (Samson et al., 2004), was used as the outgroup
species. Alignments are available on request.
Differences were observed between the topologies obtained
with the three data sets analyzed (EF-1α, TUB and PC4). Tests were
conducted to compare the topologies using the CONSEL package
version 0.li (Shimodaira and Hasegawa, 2001), which implements the
AU test (Shimodaira and Hasegawa, 2001), the KH test (Kishino and
Hasegawa, 1989), the SH test (Shimodaira and Hasegawa, 1999) and
the RELL bootstrap proportions (Shimodaira and Hasegawa, 1999).
These tests compare the p-value associated with each of several given
trees, which represents the possibility of that tree being the true tree
given the sequence data. The competing topologies are thus ranked
according to their p-values in order to determine which one is the
most likely.
3. Results
3.1. Phylogenetic analysis of Penicillium from the cheese environment
using frequently used protein coding genes
Four genes frequently used in fungal molecular taxonomy were
sequenced. The mitochondrial COI gene, advertised as a good candidate for the barcoding of Penicillium (Seifert et al., 2007), and the
calmoduline gene were both not informative, only differentiating
P. palitans from the other species of interest, i.e. P. camemberti,
P. biforme, P. fuscoglaucum, P. caseifulvum. The two other nuclear
genes, EF-1α and TUB, were informative, but the topologies obtained
from those genes were not completely congruent (Fig. 1). In the
phylogenetic tree inferred from the EF-1α dataset, three clades were
well supported. Two were sister clades, one containing P. palitans
isolates and the second including ten isolates morphologically identified as P. commune, one of which being the ex-type of P. fuscoglaucum
(NRRL 892 = LCP 91.2798), a synonym of P. commune. The third major
208
F. Giraud et al. / International Journal of Food Microbiology 137 (2010) 204–213
Fig. 1. Maximum likelihood phylogeny inferred from the β-tubulin (A) and translation elongation factor (B) datasets. Phylogeny was rooted by P. crustosum. Numbers at major nodes
indicate percent ML bootstrap values from 1000 bootstrapped datasets. Branch lengths are proportional to the inferred amount of evolutionary change and the scale represents 100
nucleotide substitutions per site.
clade grouped P. camemberti isolates (including old synonyms of
the species, i.e., P. candidum, P. rogeri, P. caseicola, P. biforme),
P. caseifulvum and ten P. commune isolates, including the ex-type
strain (MUCL 34882) and the neotype strain (CBS 216.30). The TUB
phylogeny differed in (i) the sister clade of P. palitans, composed of
a different set of P. commune isolates, including the ex-type of
P. biforme, and (ii) the major clade, which thus encompassed the
isolates that belonged to the second clade in the EF-1α tree (including
F. Giraud et al. / International Journal of Food Microbiology 137 (2010) 204–213
P. fuscoglaucum ex-type). Summaries of the alignments for these
genes are shown in Table 4. Variable positions (parsimony informative) among ingroup species are detailed in Table 5.
209
Table 5
Nucleotide variability observed within the β-tubulin and the translation elongation
factor genes between the four ingroup species (P. ca: P. camemberti, P. bi: P. biforme, P.
fu: P. fuscoglaucum, P. pa: P. palitans).
Variable positions
(parsimony informative)
on the EF-1α alignment
Species Isolates Variable positions
number (parsimony informative)
on the TUB alignment
3.2. Microsatellite variations between contaminant species and
P. camemberti
Out of the 23 primer pairs designed to amplify microsatellite
loci, 17 successfully amplified fragments of expected size (Table 3).
Six primer pairs designed to amplify microsatellite regions from
P. camemberti were tested for their discrimination ability of contaminant species closely related to P. camemberti (Table 1), both using
size of the amplification and substitutions in the flanking regions.
We choose PC4 for subsequent analyses because it gave readable
sequences for most of the strains and appeared to differentiate the
species.
The PC4 marker is a small fragment whose size ranged from 149 bp
(P. crustosum, outgroup species) to 169 bp (P. camemberti). Summary
of the alignment is shown in Table 4. Variations among ingroup
species were observed both in the number of AG repeats (from 9 to
12) and as substitutions in the nonrepetitive regions flanking the
microsatellite (Table 6).
Seven haplotypes were observed among the 52 isolates studied,
segregating in 4 clades in the phylogenies. The ML and most
parsimonious trees, constructed considering gaps as missing characters (ML) or as a fifth character (which allow to include variations in
the repeat numbers in the parsimony analysis), showed identical
topologies (ML tree shown in Fig. 2). Three clades had 100% bootstrap
support and each harboured identical haplotypes (description in
Table 6). One of the clades corresponded to P. palitans, showing an
interruption of the microsatellite AG repeat at position 122 of the
alignment due to a G to A transition. A second clade corresponded to
P. biforme (placed in synonymy with P. camemberti by Pitt in, 1979),
identified based on the sequence of the ex-type isolate CBS 297.48,
and included 9 isolates deposited in collections as P. commune. A third
clade included P. camemberti isolates (including ex-type isolates
bearing synonymized names : P. candidum, P. rogeri, P. caseicola), with
the ex-type (MUCL 34882 = NRRL 890) and the neotype (designated
by Pitt et al., 2001 as CBS 216.30, T of P. lanosogriseum) isolates of
P. commune, and with isolates of P. caseifulvum. In the fourth clade, all
the isolates shared the same substitutions in positions 30, 80, 156
and 173 of the alignment, but differed at the position 94 (A/G) and in
the number of AG repeats, resulting in four haplotypes: the ex-type
isolate of P. fuscoglaucum (NRRL 892 = CBS 261.69 = MUCL 28651)
and LCP 07.5471 carried a G in position 94, as did all the isolates
outside of this clade, and shared 12 AG repeats with P. camemberti,
while the remaining isolates of the clade had a substitution in position
94 (A instead of G) and 10 AG repeats, with the exception of LCP
07.5472 (9 AG repeats) and CMPG74 (11 AG repeats). This clade
included P. fuscoglaucum ex type isolate (regarded as a possible synonym of P. commune by Raper and Thom, 1949 and Samson et al,
1976) and 9 isolates previously identified as P. commune.
Thus, from the 22 isolates received from culture collections as
P. commune, none showed the sequences of the ex-type (and neotype)
P.
P.
P.
P.
ca
bi
fu
pa
114
G
G
G
T
22
11
11
6
128
T
C
T
C
223
C
T
C
C
230
C
C
C
T
264
C
C
C
T
301
G
G/A
G
G
184
T
T
C
C
185
T
T
C
C
188
C
C
A
A
225
T
T
A
A
229
T
T
T
C
isolate of the species. They corresponded instead to the old species
P. fuscoglaucum and P. biforme. Furthermore, the ex-type isolate and
the neotype of P. commune were strictly identical to P. camemberti at
all examined sequences.
3.3. Results of enforced topologies
The individual gene trees reconstructed for PC4, TUB and EF-1α had
incongruent topologies, with four clades in the PC4 tree and only three
clades in the TUB and EF-1α trees. The genealogy of PC4 supported the
four species P. biforme, P. camemberti (sensu lato), P. fuscoglaucum and
P. palitans, while TUB distinguished P. biforme and P. palitans from a
main clade encompassing P. camemberti and P. fuscoglaucum. EF-1α in
contrast distinguished P. fuscoglaucum and P. palitans from a main
clade encompassing P. camemberti and P. biforme. We tested whether
the sequences obtained for EF-1α and TUB could be consistent with
the PC4 topology using the enforced topology test (CONSEL analysis,
supplementary data). In the case of EF-1α, the estimated ML phylogenetic tree was ranked as the most likely in all comparisons,
indicating that the topology supported by the EF-1α sequences is
significantly different from that supported by PC4. In the case of TUB,
the enforced topology was ranked first in all tests, indicating that the
topology supported by the TUB sequences is not significantly different
from that supported by PC4.
3.4. Variations and cross-amplifications obtained for microsatellite loci
screened on the set of biotechnological Penicillium isolates
Out of the 17 primer pairs designed to amplify microsatellite loci
that successfully yielded fragments of expected size in the species
where they were developed, nine cross-amplified in other Penicillium
species (Table 2). None of them differentiated P. chrysogenum from
P. nalgiovense. Six markers can be used for differentiating the starters
at the species level (PC1, PC2, PC11, PC13, PR6, and PR7). Five loci
from the PC library amplified specifically in P. camemberti (PC4, PC5,
PC6, PC7, and PC12). Most of the markers appeared monomorphic
within the species tested. PR4Bis and PR5 showed some variability in
size amplification respectively in both P. camemberti and P. roqueforti.
Table 6
Variations observed within the locus PC4 between the four ingroup species.
Table 4
Characters of each DNA sequence alignment.
Species
DNA region
TUB
EF-1α
PC4
Length of the final alignment
Constant characters
Variable characters
Informative characters
Mean ingroup sequence divergence (p-distance %)a
382
359
21
16
0.8
566
551
15
15
0.6
173
152
21
19
2
a
Ingroup species: P. biforme, P. camemberti, P. fuscoglaucum, P. palitans.
P.
P.
P.
P.
ca
bi
fu
pa
Variable positions on the nonrepetitive regions
flanking the microsatellite
30
T
T
C
T
80
A
A
G
A
94
G
G
A/G
G
102
C
C
C
T
122
G
G
G
A
156
T
C
C
T
P. ca: P. camemberti, P. bi: P. biforme, P. fu: P. fuscoglaucum,
P. pa: P. palitans.
AG repeats
173
T
C
C
T
134/139
12
9
9 to 12
3+6
210
F. Giraud et al. / International Journal of Food Microbiology 137 (2010) 204–213
Fig. 2. Maximum likelihood phylogeny inferred from the microsatellite locus PC4. Phylogeny was rooted by P. crustosum. Numbers at major nodes indicate percent ML bootstrap
values from 1000 bootstrapped datasets. Branch lengths are proportional to the inferred amount of evolutionary change and the scale represents 10 nucleotide substitutions per site.
3.5. Phenotypical analysis of P. commune isolates
Growth abilities of isolates identified as P. commune were
measured (Table 7), taking representatives of the different clades
where the P. commune morphospecies was found in phylogenies. The
optimal temperature for growth on CYA was 25 °C for P. commune
isolates belonging to the P. fuscoglaucum clade and for the ex-type
strain of P. commune, while P. commune isolates belonging to the
P. biforme clade showed an optimal range for growth comprised
between 15 °C and 25 °C. The mean diameters at the optimal temperature were 35 mm for the ex-type strain of P. commune, 33 mm for
the P. commune belonging to the P. fuscoglaucum clade and 25–26 mm
for the P. commune belonging to the P. biforme clade. The ex-type
strain of P. commune showed a reduced growth at 10 °C (15 mm) in
comparison to the other strains under study (22 mm). No growth was
observed at 37 °C. The colour of the colonies and of their reverse was
Table 7
Growth ranges (mm) of isolates on MEA and CYA incubated at different temperatures.
Species
MEA 25 °C
CYA 10 °C
CYA 15 °C
CYA 25 °C
CYA 30 °C
P. fu
P. bi
P. coa
(28)a 25–35
(26) 20–30
30
(22) 18–24
(22) 19–25
15
(26) 17–30
(25) 20–30
25
(33) 25–40
(26) 20–30
35
(17) 12–22
(16) 12–20
17
a
Mean values; P. co ex-type strain MUCL34882.
F. Giraud et al. / International Journal of Food Microbiology 137 (2010) 204–213
not significantly different from one clade to the other, nor was the
microscopic aspect of the conidial structures.
4. Discussion
To study fungi from the cheese environment, there is a crucial need
for both strain typing and species recognition, as starters and
contaminants are very closely related. In particular, P. camemberti is
possibly derived from the contaminant P. commune, and P. roqueforti,
the starter for Roquefort cheese, is occurring as a contaminant in other
hard cheeses. In the present study, the usefulness of microsatellite
markers to discriminate domesticated fungi of the genus Penicillium
was therefore explored.
Few studies have used microsatellites for species recognition so
far, and they focused on cryptic species in pathogenic fungi (Fisher
et al., 2000; Pringle et al., 2005; Matute et al., 2006; Giraud et al.,
2008c). Microsatellites have been more commonly used to investigate
intraspecific variability for population genetics studies (Giraud, 2004;
Tuthill, 2004; Lopez-Villavicencio et al., 2007; Giraud et al., 2008b)
and epidemiology (Taylor and Fischer, 2003).
Polymorphic microsatellites are often difficult to isolate in fungi,
because of their scarcity and shortness in most genomes (Dutech
et al., 2007). We in fact had to build three enriched libraries in
P. roqueforti in order to find suitable loci, with at least six repeat units,
a common threshold for variation (Dettman and Taylor, 2004). We
finally could obtain several loci from P. camemberti and P. roqueforti
libraries yielding good amplifications and appearing as good candidates for taxonomy purpose. Six loci were able to discriminate species,
with one specific allele for each of the biotechnological species,
P. camemberti, P. roqueforti and P. nalgiovense. Five loci amplified only
P. camemberti. No locus was able to discriminate P. chrysogenum
starter isolates from P. nalgiovense. Using southern blots of RAPD PCR
products we have previously shown that P. chrysogenum starters
belonged in fact to P. nalgiovense (Dupont et al., 1999).
We explored the sequences of the flanking regions of microsatellite loci, in comparison to four gene sequences, to assess their
utility for investigating the origin of P. camemberti among the closely
related cheese contaminant species. P. camemberti is considered as a
domesticated species (Samson and Frisvad, 2004) and its ancestral
wild type was thought to be P. commune (Pitt et al., 1986), originally
isolated from cheese (Thom, 1910). Both antigenic characterization
(Polonelli et al., 1987) and molecular data (Samson et al., 2004)
supported this genealogy. Here, we showed that the ex-type isolate
of P. commune (NRRL 890 = MUCL 34882) was genetically strictly
identical to P. camemberti at the five DNA fragments examined.
Moreover, none of the other twenty isolates that we received as
P. commune shared the ex-type isolate genotype. They were instead
distributed into two lineages which were identified as P. fuscoglaucum
and P. biforme with reference to the ex-type isolates of these old
synonyms. These results suggest that P. commune may not exist apart
from the ex-type isolate and that P. camemberti could be a color
mutant of P. commune. The history of cheese molds began in northern France a long time before Thom published the description of
P. camemberti (1906) and P. commune (1910) from imported Camembert, using earlier names such as P. album, P. rogeri, P. candidum
and P. caseicola (see Thom, 1930 for a review). The ex-type isolates of
these old species were shown here to be identical to P. camemberti, to
which they have been synonymysed (Raper and Thom, 1949; Samson
et al., 1977). P. camemberti is also indistinguishable from P. caseifulvum, recently described to accomodate non-cyclopiazonic acid
producing strains isolated from Danish blue and other German and
French cheeses, and proposed as a candidate for fermenting cheeses
or salami (Lund et al., 1998). There is no status for domesticated
species in the International Code for Botanical Nomenclature. Despite
the apparent nomenclatural problem of P. commune and P. camemberti, it seems unrealistic to make any name changes for such
211
important industrial species, as recommended for other important
Aspergillus and Penicillium (Frisvad et al., 1990).
Using a significant number of isolates and multiple gene
genealogies, we showed the existence of two divergent clades in the
morphological species P. commune, both genetically distinct from
the ex-type strain. According to the GCPSR criterion, a phylogenetic
species is recognized if it appears as a well-supported clade in the
majority of single-locus genealogies and is not contradicted by any
single-locus genealogy (Taylor et al., 2000). In this study, the gene
trees obtained from TUB, EF-1α and PC4 were not concordant in the
number of clades resolved among the P. commune isolates, but there
was no contradiction in the isolate composition of the clades. Two
clades were delineated within the phylogeny based on the microsatellite locus PC4, identified as P. fuscoglaucum and P. biforme with
reference to the ex-type strains of these old species. Either one clade
or the other was resolved from EF-1α (P. fuscoglaucum) and TUB
(P. biforme), the remaining isolates being indistinguishable from
P. camemberti. To check if the TUB and EF-1α data significantly
supported different topologies than the one obtained using PC4, we
enforced the topologies from those datasets to fit with the two clades
obtained with PC4. The TUB dataset could be consistent with the PC4
topology. Moreover, growth characters supported the distinct clades.
Higher growth diameters were observed for the isolates of the
P. fuscoglaucum clade and a better ability to grow at low temperatures
characterized the isolates of the P. biforme clade. According to these
results and to the industrial context of this study, where workers
need to recognize accurately spoiler fungi for a safe management of
the processes, we finally decided to recognize two genealogical species. We proposed to re-introduce the old names P. fuscoglaucum
(synonymised with P. commune) and P. biforme (synonymised with
P. camemberti), as the ex-type strains of these species were
representative of the two divergent clades obtained and for which a
legitimate status was recognized in the fungal nomenclature database
MYCOBANK (http://www.mycobank.org). P. biforme and P. fuscoglaucum are undoubtedly very closely related to P. camemberti. From the
phylogenies, the P. biforme clade contained mainly isolates from the
cheese environment, while the P. fuscoglaucum clade contained
isolates from various substrates. P. palitans could be a more generalist
food contaminant (as mentioned in Frisvad and Samson, 2004). Most
of the isolates deposited in collections have however been randomly
collected and it is extremely difficult to attest their real origin, as they
could be migrants from other environments. Rational sampling in
cheese factories and in other food environments has to be performed
to better understand the ecology of the species. If some spoiler species
are really confined to the cheese environment, then they may be
considered as domesticated organisms as well, but unconsciously
adapted to this human activity (Gepts and Papa, 2002). The wild
origin of cheese fungi remains to be determined. Phylogenetic studies
could help retracing the history of divergence for some fungi. The
domesticated strains of Saccharomyces cerevisiae specialized for the
production of alcoholic beverages were shown to be derived from
natural populations found on oak exudates in North America (Fay and
Benavides, 2005). Mycosphaerella graminicola, the wheat pathogen,
diverged from an ancestral population infecting wild grasses in the
Middle East, approximately 10 500 years ago (Stukenbrock et al.,
2007). For the Basidiomycete Serpula lacrimans, the transition from
the forest to the human habitation has favoured the split of the species
into two lineages (Kauserud et al., 2004).
Among biotechnological isolates of P. camemberti and P. roqueforti
provided by starter producers, we did not found any polymorphism.
Starters may represent a single clone in each species (personal
communication from Producers), but more markers on a larger
sample including wild isolates should be typed to assess the level of
polymorphism. Although domestication is known to reduce genetic
diversity in general (Gepts and Papa, 2002) additional research should
be conducted on P. roqueforti, because it is known from other habitats
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F. Giraud et al. / International Journal of Food Microbiology 137 (2010) 204–213
than cheese, such as soil, silage and wood (Pitt and Hocking, 1999;
Samson and Frisvad, 2004; O'Brien et al., 2008).
In conclusion, this study brought several insights on cheese fungi: 1)
the molecular identity of P. camemberti isolates with the ex-type isolate of P. commune, 2) the extreme rarity of P. commune, and 3) the
identification of spoilers related to P. camemberti as P. biforme,
P. fuscoglaucum and P. palitans. This study provided several microsatellite
loci useful in strain typing and species recognition. Among them, the
PC4 locus, providing good signatures for each species of interest, might
be a useful marker to barcode these economically important species.
Acknowledgements
We thank John Taylor for critical review of the manuscript and Yves
Brygoo for useful comments. We thank Yves Brygoo, Michael Solignac,
Dominique Vautrin, Benjamin Genton, and Rumsaïs Blatrix for help
in mirosatellite development. This work was partly supported by the
“Consortium National de Recherche en Génomique”, and the “service de
systématique moléculaire” of the Muséum National d'Histoire Naturelle
(IFR 101). It is part of the agreement number 2005/67 between the
Genoscope and the Muséum National d'Histoire Naturelle on the project
“Macrophylogeny of life” directed by Guillaume Lecointre.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ijfoodmicro.2009.11.014.
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