Microsatellite loci to recognize species for the cheese starter and contaminating strains associated with cheese manufacturing

International Journal of Food Microbiology 137 (2010) 204–213

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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

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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.

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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

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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

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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.

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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

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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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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. References Barriel, V., 1994. Molecular phylogenies and how to code insertion/deletion events. Comptes Rendus de l' Académie des Sciences Paris, Series III 317, 693–701. Boysen, M., Skouboe, P., Frisvad, J., Rossen, L., 1996. Reclassification of the Penicillium roqueforti group into three species on the basis of molecular genetic and biochemical profiles. Microbiology 142, 541–549. Dettman, J.R., Jacobson, D.J., Turner, E., Pringle, A., Taylor, J.W., 2003. Reproductive isolation and phylogenetic divergence in Neurospora: comparing methods of species recognition in the model eukaryote Neurospora. Evolution 57, 2721–2741. Dettman, J.R., Taylor, J.W., 2004. Mutation and evolution of microsatellite loci in Neurospora. Genetics 168, 1231–1248. Dupont, J., Magnin, S., Marti, A., Brousse, M., 1999. Molecular tools for identification of Penicillium starter cultures used in the food industry. International Journal of Food Microbiology 49, 109–118. Dutech, C., Enjalbert, E., Fournier, E., Delmotte, F., Barrès, B., Carlier, J., Tharreau, D., Giraud, T., 2007. Challenges of microsatellite isolation in fungi. Fungal Genetics and Biology 44, 933–949. Fay, J.C., Benavides, J.A., 2005. Evidence for domesticated and wild populations of Saccharomyces cerevisiae. PLoS Genet 1, e5. Felsenstein, J., 1989. PHYLIP — Phylogeny Inference Package (Version 3.2). Cladistics 5, 164–166. Fisher, M.C., Koenig, G., White, T.J., Taylor, J.W., 2000. A test for concordance between the multilocus genealogies of genes and microsatellites in the pathogenic fungus Coccidioides immitis. Molecular Biology and Evolution 17, 1164–1174. Frisvad, J.C., Hawksworth, D.L., Kozakiewicz, Z., Pitt, J.I., Samson, R.A., Stolk, A.C., 1990. Proposals to conserve important species names in Aspergillus and Penicillium. In: Samson, R.A., Pitt, J.I. (Eds.), Modern concepts in Penicillium and Aspergillus classification. Plenum Press, New York, pp. 83–90. Frisvad, J.C., Samson, R.A., 2004. Polyphasic taxonomy of Penicillium subgenus Penicillium. A guide to identification of food and air-borne terverticillate Penicillia and their mycotoxins. Studies in Mycology 49, 1–173. Geisen, R., Cantor, M.D., Hansen, T.K., Holzapfel, W.H., Jakobsen, M., 2001. Characterization of Penicillium roqueforti strains used as cheese starter cultures by RAPD typing. International Journal of Food Microbiology 65, 183–191. Geiser, D.M., Harbinski, F.M., Taylor, J.W., 2000. Molecular and analytical tools for characterizing Aspergillus and Penicillium species at the intra- and interspecific levels. In: Samson, R.A., Pitt, J.I. (Eds.), Integration of modern taxonomic methods for Penicillium and Aspergillus classification. Harwood Academic Publishers, Amsterdam, The Netherlands, pp. 381–394. Gepts, P., Papa, R., 2002. Evolution during domestication. Encyclopedia of Life Sciences. Nature Publishing group, London. Glass, N.L., Donaldson, G.C., 1995. Development of primer sets designed for use with PCR to amplify PCR conserved genes from filamentous Ascomycetes. Applied and Environmental Microbiology 61, 1323–1330. Giraud, T., Fournier, E., Vautrin, D., Solignac, M., Vercken, E., Brygoo, Y., 2002. Isolation of eight polymorphic microsatellite loci, using an enrichment protocol, in the phytopathogenic fungus Fusarium culmorum. Molecular Ecology Notes 2, 121–123.

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