Epicoccum sp. as the causative agent of a reddish-brown spot defect on the surface of a hard cheese made of raw ewe milk

In e na ional Jou nal o Food Mic obiology 406 (2023) 110401 A ailable online 16 Sep embe 2023 0168-1605/© 2023 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/). Epicoccum sp. as he causa i e agen o a eddish-b own spo de ec on he su ace o a ha d cheese made o aw ewe milk Ja ie Rod íguez, Lucía V´ azquez, Ana Bel´ en Fl´ o ez, Bal asa Mayo * Depa amen o de Mic obiología y Bioquímica, Ins i u o de P oduc os L´ ac eos de As u ias (IPLA), Consejo Supe io de In es igaciones Cien í icas (CSIC), Paseo Río Lina es s/n, 33300 Villa iciosa, Spain Ins i u o de In es igaci´ on Sani a ia del P incipado de As u ias (ISPA), A enida de Roma s/n, 33011 O iedo, Spain ARTICLE INFO Keywo ds: Epicoccum Moulds Fungi Cheese de ec Colou de ec Cheese mycobio a Cheese mic obio a ABSTRACT Colou de ec s can a ec he appea ance o cheese, i s la ou , he sa e y o i s consump ion, and he p ice i can demand. This wo k epo s he iden i ica ion o i e ungal isola es om a dai y plan whe e he su ace o mos cheeses was a ec ed by pa en , eddish- o-b own s ains. One o hese isola es was ob ained om cheese, wo om b ine, and wo om a bulk ank con aining ewe milk. Molecula iden i ica ion by pa ial ampli ica ion, sequencing, and da abase compa ison o he conca ena ed sequence o he genes coding o he la ges subuni o RNA polyme ase II (RPB2), β- ubulin (β-TUB), and he la ge subuni o he RNA molecule (LSU), plus he in- e nal ansc ibed sequence (ITS) egions, assigned he isola es o Epicoccum layuense, Epicoccum i alicum, and Epicoccum mezze ii. Fea u es o he g ow h o hese di e en species on di e en aga -based media, and o he mo phology o hei conidia ollowing spo ula ion, a e also epo ed. The s ain isola ed om cheese, E. layuense IPLA 35011, was able o ec ea e he eddish-b own s ains on slices o Gouda-like cheese, which linked he ungus wi h he colou de ec . In addi ion, wo o he s ains, E. i alicum IPLA 35013 om b ine and E. i alicum IPLA 35014 om milk, also p oduced s ains on cheese slices. Epicoccum species a e widely ecognized as plan pa hogens bu ha e seldom been epo ed in he dai y se ing, and ne e as human o animal pa hogens. 1. In oduc ion The mic obio a o cheese comp ises a as a ay o p oka yo ic and euka yo ic o ganisms and hei i uses (Mayo e al., 2021). Cheese- associa ed euka yo es include bo h yeas s and ilamen ous ungi. The majo i y o yeas species belong o he gene a Geo ichum, Deba yomyces, Kluy e omyces, Candida, and Ya owia (Bin sis, 2021; F ¨ ohlich-Wyde e al., 2019), while he dominan ilamen ous ungi belong o Penicillium species, such as P. camembe i, P. oque o i, and P. commune, e c. (Ropa s e al., 2020). O he ilamen ous ungi o he gene a Fusa ium and Sco- pula iopsis may also be sp ead h oughou cheeses, bu in low numbe s (Cenci-Goga e al., 2021; Moubashe e al., 2018). Via hei complex enzyme sys ems, some o hese o ganisms con ibu e o he biochemical eac ions o ipening (p o ein b eakdown, a hyd olysis, and/o lac ose me abolism) in ce ain cheese a ie ies, helping o p oduce hei ypical appea ance, as e, and a oma (A sha i e al., 2020; Gi a a, 2021). Howe e , unwan ed ungal de elopmen can occu , leading o quali y de ec s and e en ood sa e y issues by he p oduc ion o myco oxins (Ri scha d e al., 2018). The mos common cheese-spoiling de ec s include discolo a ions, bi e ness, c ys al o ma ion, and an open (spli ) ex u e (Aga wal e al., 2006; Bassi e al., 2015; Kamelamela e al., 2018). These migh ha e di e en causes and can be modula ed by in insic and ex insic ac o s, such as empe a u e, pH, humidi y, calcium o p o ein con en , s o age condi ions, e c. Colou de ec s a e usually caused by he accumula ion o high concen a ions o mic obial pigmen s. A ypically colou ed cheeses a e equen ly epo ed by bo h he indus ial and small-scale cheese p oduc ion sec o s (Daly e al., 2012), and commonly en ail p oblems o consume accep ance and economic p o i abili y. Li le is known abou hese pigmen s o he o ganisms ha p oduce hem, al hough ecen ly, species o P o eus and Psych obac e ha e been associa ed wi h a pu ple colou de ec (caused by indigo and indi ubin p oduc ion om yp o- phan) in a su ace- ipened cheese (Kamelamela e al., 2018). Simila ly, The mus he mophilus isola ed om a pink discolou a ion de ec in a Con inen al-cheese ype was shown o p oduce lycopene, a ed ca o - enoid (Quigley e al., 2016). The la e au ho s we e able o ec ea e his colou de ec using he T. he mophilus isola e in a cheese model, un- equi ocally linking he o ganism wi h he de ec . In addi ion, an * Co esponding au ho a : Ins i u o de P oduc os L´ ac eos de As u ias (IPLA-CSIC), Paseo Río Lina es s/n, 33300 Villa iciosa, Spain. E-mail add ess: [email p o ec ed] (B. Mayo). Con en s lis s a ailable a ScienceDi ec In e na ional Jou nal o Food Mic obiology jou nal homepage: www.else ie .com/loca e/ij oodmic o h ps://doi.o g/10.1016/j.ij oodmic o.2023.110401 Recei ed 30 Ma ch 2023; Recei ed in e ised o m 29 June 2023; Accep ed 13 Sep embe 2023 In e na ional Jou nal o Food Mic obiology 406 (2023) 110401 2 in e ac ion be ween Glu amicibac e a ilai ensis (a bac e ium) and Peni- cillium species ( ungi) has been shown o cause an o e p oduc ion o cop opo phy in III by he o me o ganism (Clea y e al., 2018). This pigmen has been iden i ied as he cause o a pink ma bling de ec on he su ace o some smea - ipened aged cheeses. Al hough bac e ia a e he cause o mos de ec s, moulds g owing on he su ace o non-mouldy cheeses, o he de elopmen o non-s a e s ains inside o on he ind o mouldy cheese a ie ies, can cause aes he ic al e a ions and p oduce o - la ou s (Daly e al., 2012; Fe ocino e al., 2022). The o igin o con aminan moulds may be he s a ing milk, o he ing edien s ( enne , calcium chlo ide, colo an s, sal , e c.), o he manu ac u ing and ipening en i onmen s (Ga nie e al., 2017). This wo k epo s a ungus isola ed om he su ace o a Manchego- ype ha d cheese made om aw ewe milk in cen al Spain (Cas ile- Leon), ha caused s iking, ed-b own spo s on he cheese ind. Sam- pling pe o med in he cheesemaking ac o y iden i ied u he moulds o he same genus in he bulk ank milk and on equipmen and ools in he manu ac u ing and ipening en i onmen s. Molecula iden i ica ion assigned he isola es o di e en species o Epicoccum, a ungal genus associa ed wi h spo s on he lea es o di e en plan species, bu a ely epo ed in cheese. 2. Ma e ial and me hods 2.1. Cheese sampling and cul u e condi ions Colou ed pa ches o he cheese su ace we e sampled wi h s e ile scalpels, and he ma e ial ob ained was inocula ed di ec ly on aga pla es o suspended in Ringe ’s solu ion (Me ck, Da ms ad , Ge many) o p epa e en- old dilu ions. Fungi we e isola ed on pla e coun aga wi h skimmed milk (PCAM). Isola es we e pu i ied and ou inely g own on he same medium o on yeas -ex ac glucose chlo amphenicol (YGC) aga pla es (bo h media om Me ck). Hyphae om moulds on he cheese su ace we e eco e ed in co on swabs and inocula ed di ec ly in o YGC pla es. As con ols, samples we e aken and ea ed in he same way om non-colou ed and non-mouldy a eas o he cheese su ace. 2.2. DNA ex ac ion and pu i ica ion Fungal genomic DNA was ex ac ed om he mycelium a e g ow h on YGC aga pla es. Ex ac ion and pu i ica ion we e pe o med using he QIAmp DNA Mini Ki (Qiagen, Hilden, Ge many) ollowing he manu ac u e ’s ins uc ions wi h mino modi ica ions. In sho , cells we e lysed in 1.5 mL o lysis bu e con aining 200 mM T is-HCl, 2 mM EDTA, 2 mM NaCl, and 500 mM SDS. The mix u e was hen placed in a sc ew-cap ube con aining 0.15 g o bo h 0.1 and 0.5 mm zi conia/silica beads (BioSpec P oduc s, Ba les ille, OK, USA) and subjec ed o me- chanical b eakage in a Fas P ep FP120 Cell Dis up e (Qbiogene, Ca lsbad, CA, USA) o h ee cycles a 5.5 m s −1 o 40 s, wi h cooling on ice o 30 s be ween cycles. This ly ic suspension was used o con inue wi h he DNA ex ac ion using he QIAmp DNA Mini ki . Finally, he DNA was elu ed wi h 100 μ L o molecula g ade wa e (Sigma-Ald ich, S . Louis, CA, USA) and s o ed a −20 ◦C un il analysis. 2.3. Molecula iden i ica ion Molecula iden i ica ion was achie ed by ampli ica ion, sequencing, and compa ison o conca ena ed sequences om ou genomic egions agains hose held in da abases. Ribosomal in e nal ansc ibed se- quences (ITS) we e ampli ied by PCR using p ime s ITS1-F (5ʼ- TCCGTAGGTGAACCTGCGG-3ʼ) and ITS4-R (5ʼ-TCCTCCGCTTATTGA- TATGC-3ʼ), as epo ed by Kageyama e al. (1997). Pa ial ampli ica ion o he gene coding o he la ges subuni o RNA polyme ase II (RPB2) was pe o med using PCR wi h p ime s RPB2-RF2 (5 ′ -GGGGWGAYCA- GAAGAAGGC-3 ′ ) (Sung e al., 2007) and RPB2-7C (5 ′ - CCCATRGCTTGTYYRCCCAT-3 ′ ) (Liu e al., 1999). Pa ial ampli ica ion o he genes coding o β- ubulin was pe o med using he p ime s B ub2Fd (5 ′ -GTBCACCTYCARACCGGYCARTG-3 ′ ) B ub4Rd (5 ′ - CCRGAYTGRCCRAARACRAAGTTGTC-3 ′ ) (Woudenbe g e al., 2009). Finally, pa ial ampli ica ion o he la ge subuni o he RNA molecule (LSU) was accomplished wi h p ime s LR0R (5 ′ -GTACCCGCTGAACT- TAAGC-3 ′ ) and LR5 (5 ′ -ATCCTGAGGGAAACTTC-3 ′ ) (Vilgalys and Hes- e , 1990). Ampli ica ion condi ions we e as epo ed in he espec i e e e ed a icles. Amplicons we e isualized ia aga ose (1 %) gel elec opho esis, pu i ied, and sequenced a Mac ogen (Mad id, Spain). Sequences we e analysed using Ch omas so wa e (Technelysium, Sou h B isbane, Aus alia) and compa ed wi h hose o e e ence axa in NCBI GenBank using he BLAST p og am (h ps://blas .ncbi.nlm.nih.go /Bla s .cgi). 2.4. Phylogene ic analyses App op ia e axa o he phylogene ic analysis we e ini ially selec ed ollowing BLAST sea ches o he GenBank da abase. Rep esen a i e DNA sequences om Epicoccum species we e selec ed and downloaded o be used as a e e ence in he phylogene ic analysis. Supplemen a y Table 1 summa ises he lis o species, s ains, sou ces, geog aphical o igins and GenBank accession numbe s o he e e ence specimens consul ed. Alignmen s o consensus sequences o he isola es and e e ence se- quences we e pe o med using Molecula E olu iona y Gene ics Anal- ysis (MEGA) .11.0 so wa e (Tamu a e al., 2021), using s anda d se ings and employing he MUSCLE algo i hm. When necessa y, alignmen s o ou isola es we e isually inspec ed and he sequences co ec ed manually. Finally, phylogene ic ela ionships o conca ena ed sequences o he di e en alignmen s we e de e mined ollowing he ecommenda ions by Chai e al. (2019) using he maximum composi e likelihood algo i hm. 2.5. Mo phology and pheno ypic cha ac e iza ion The mo phological and cul u al cha ac e is ics o he iden i ied s ains we e ca e ully examined on mal ex ac aga (MEA) (Scha lab, Ba celona, Spain), oa aga (OA) (Me ck), po a o dex ose aga (PDA) (Me ck), and CZAPEK aga (Me ck), acco ding o he ecen ecom- menda ions o El-Sayed e al. (2020). Unless o he wise s a ed, all cul- u es we e g own a 20 ◦C o 5–15 days. To allow o spo ula ion, he s ains we e incuba ed u he a 25 ◦C unde cycles o 12 h ul a iole ligh and 12 h comple e da kness o 3 ex a days. Conidia we e obse ed using a Leica DMi8 in e ed mic oscope (Leica, We zla , Ge many) and pho og aphed unde b igh ield illumina ion using a Leica DFC 365FX came a. 2.6. Fungal de elopmen in cheese slices Slices o a comme cial Gouda-like cheese we e exposed o UV ligh o 15 min in a labo a o y cabine . Slices we e hen insed wi h s e ile wa e and placed on op o mois ened il e pape s on a la ge Pe i dish. Finally, mould s ains we e inocula ed on he cen e o he cheese slice and incuba ed a 15–18 ◦C o up o 10 days. 2.7. Accession numbe s The DNA sequences ob ained in his s udy o he ITS egions, and he LSU, β-TUB, and RPB2 genes om he IPLA 35011, IPLA 35013, IPLA 35014, IPLA 35015, and IPLA 35017 s ains we e deposi ed in GenBank da abase unde accession numbe s OQ568299-OQ568303, OQ570976- OQ570980, OQ599322-OQ599326, and OQ605790-OQ605794, espec i ely. 3. Resul s The su ace o he cheeses a he ac o y de eloped pa en eddish- J. Rod íguez e al. In e na ional Jou nal o Food Mic obiology 406 (2023) 110401 3 b own spo s while ipening in he cold oom (Fig. 1, A1). Occasionally, hese spo s lay unde a laye o whi e mould mycelium (Fig. 1, A2–A3) and we e only isible a e emo ing his coa ing. De elopmen o he spo s was usually s onge on cheeses ou o he way o ai cu en s gene a ed by he e ige a o s. They occupied a hin laye on he su ace o he ind wi hou en e ing he cheese ma ix. The cheeses a e b ushed wi h oli e oil be o e being sen o ma ke , a p ocess ha hid he spo s and would ha e sa egua ded agains appea ance de ec s and nega i e pu chase a i udes. Howe e , he company wan ed o iden i y he cause o he con amina ion in case o ood sa e y issues. Small sec ions o he spo s on he su ace o a ma u e cheese we e emo ed using a s e ile scalpel, and cul u ed on PCAM aga pla es. A ce ain loca ions on he e e se o some pla es, eddish-b own s ains we e seen agains a backg ound yeas -like lawn (Fig. 1, B). Sepa a e yeas -like and ilamen ous ungus-like colonies we e pu i ied on YGC aga pla es, bu only he la e ype p oduced colou ed spo s simila o hose seen on he cheeses (Fig. 1, C). Ampli ica ion, sequencing, and da abase compa ison o he ibosomal ITS egions iden i ied he yeas - o m isola e (IPLA 34018) as Deba yomyces hansenii, and ha o he ilamen ous o m (IPLA 35011) as Epicoccum sp. The de elopmen o ungal mycelium by Epicoccum was ma kedly educed when (and hus he ed colou o he spo s mo e pa en ) in he p esence o D. hansenii (Fig. 1, D). This sugges s ha he spo s on he cheeses wi h no isible mycelium we e o med on op o a s ong D. hansenii backg ound, which ag ees wi h he la ge numbe o yeas colonies on he isola ion pla es. Associa ion o he colou ed spo s wi h Epicoccum was demons a ed by inocula ion o comme cial Gouda-like cheese slices wi h his s ain, which ec ea ed he eddish-b own pa ches a e 8–10 days o incuba- ion a oom empe a u e (Fig. 1, E1–E2). To assess he coloniza ion and sp eading po en ial o he ilamen ous Epicoccum s ain, sys ema ic sc eening was pe o med in he ac o y’s manu ac u ing (milk and milk anks, cheesemaking cubes, s aine , cheese clo hs, p esses, b ines, e c.) and ipening (cheeses, boxes, cold oom walls, oli e oil, e c.) en i onmen s. Twen y-se en samples we e aken di ec ly om he su ace o he cheeses, bu no moulds g ew on YGC aga pla es when inocula ed wi h mycelium om spo samples. This sugges s poo egene a ion o he (young) hyphae o his ungus. Among he yeas s and moulds g owing on he pla es om he sc eening o he ac o y’s equipmen , only ou colonies wi h an Epicoccum-like mo phology we e eco e ed, wo om b ine (IPLA 35013 and IPLA A1 A2 A3 CD B E1 E2 Fig. 1. Composi e pho og aphy showing cheeses wi h he eddish-b own colou de ec (A1–A3), and di e en pha- ses o he isola ion o specimen IPLA 35011: de ec ion o a eddish-b own agglome a e on Yeas -ex ac Chlo am- phenicol Glucose (YCG) aga pla es (B), g ow h o pu i- ied colonies o he mould and yeas on YGC pla es (C), and c oss g ow h on YGC aga o IPLA 35011 and Deba yomyces hansenii IPLA 34018 (D). De elopmen and colou o ma ion o IPLA 35011 (E1) and IPLA 35013 (E2) on Gouda-like cheese slices. J. Rod íguez e al. In e na ional Jou nal o Food Mic obiology 406 (2023) 110401 4 35017) and wo om bulk ank ewe milk samples (IPLA 35014 and IPLA 35015). None o hese, howe e , ga e he exac colou one p oduced on he pla es by he isola e om cheese IPLA 35011 on YGC aga . As IPLA 35011, s ains IPLA 35013 and IPLA 35014 also p oduced on cheese slices b own spo s simila o hose seen on cheese. Pu i ied DNA om all isola es was used as a empla e o he ampli ica ion and sequencing o ou genome segmen s (ITS, RBP2, LSU, and β-TUB). The sequences ob ained we e conca ena ed and compa ed o hose o Epicoccum species in da abases. The inal conca ena ed alignmen con ained 12 ing oup axa (E. nig um wi h wo membe s) wi h a o al o 1834 nucleo ides including gaps (599 o RPB2, 474 o ITS, 473 nucleo ides o LSU, and 288 o β-TUB). The phylogene ic clus- e ing indica ed he isola es belonged o h ee di e en Epicoccum spe- cies: E. layuense (IPLA 35011; isola ed om cheese), E. i alicum (IPLA 35013 and IPLA 35014, isola ed om b ine and ewe milk, espec i ely), and E. mezze ii (IPLA 35015 and IPLA 35017; isola ed om ewe milk and b ine, espec i ely) (Fig. 2). Al hough he use o mo phology o iden i y ungi may, in he pas , ha e esul ed in he desc ip ion o an excessi e numbe o species wi h ew di e en ial cha ac e is ics (Hibbe e al., 2007), mo phology da a a e s ill used o cha ac e ize new isola es. The de ec ed s ains we e hus inocula ed in o he cen e o OA, PDA, MEA, and CZAPEK aga pla es, and hei de elopmen a 20 ◦C was eco ded daily. Fig. 3 shows he appea ance o he colonies a e 10 days o incuba ion. No able di e - ences in he colou o he mycelium, as seen om unde and abo e he aga pla es, we e eco ded o he di e en s ains on hese media, anging om whi e o black on he op side, and om b own o black on he bo om. Colony size, mo phology, and o e all appea ance we e also highly a iable. Mo pho ype did no co ela e wi h species; indeed, la ge di e ences we e seen on some media be ween IPLA 35013 and IPLA 25014 (bo h E. i alicum), and be ween IPLA 35015 and IPLA 35017 (bo h E. mezze ii). Epicoccum isola es we e g own on YGC pla es and hen subjec ed o al e na e UV ligh and da kness cycles o o ce spo ula ion and cha ac- e ize he spo es (clamydospo es). All isola es bu one (IPLA 35017) p oduced isible black acc e ions on op o he colonies, and conidia wi h spo es could be obse ed unde he mic oscope (Fig. 4). The con- idia o all isola es looked simila , i.e., sphe ical-globose, e ucose, da k b own, wi h a basal cell, and o a diame e o 12.5–28 μ m. The appea ance and size o he spo es ag ee well wi h da a epo ed in he li e a u e o Epicoccum species (Jayasi i e al., 2017). Fig. 2. Phylogene ic ela ionships o he i e ungal isola es o his s udy (highligh ed in blue) and Epicoccum specimens o 13 s ains ep esen ing 12 ing oup axa o he genus Epicoccum. The phylogene ic ee was in e - ed om he analyses o conca ena ed align- men s o LSU, RPB2, ITS and β-TUB sequences by he Maximum Pa simony me hod. The pe cen age o eplica e ees in which he associa ed axa clus e ed oge he in he boo s ap es (1000 eplica es) a e shown nex o he b anches. J. Rod íguez e al. In e na ional Jou nal o Food Mic obiology 406 (2023) 110401 5 CZAPEK OA PDA MEA IPLA 35011 IPLA 35013 IPLA 35014 IPLA 35015 IPLA 35017 Fig. 3. Colony mo phology om on (le ) and e e se ( igh ) sides o he pla es o he i e Epicoccum s ains iden i ied in his s udy g own on pla es o Oa Aga (OA), Mal Ex ac Aga (MEA), Po a o Dex ose Aga (PDA) and CZAPEK aga , a 20 ◦C o 5 o 15 days. Fig. 4. Op ical mic oscopy o ungal spo es om ou ou o he i e Epicoccum specimens iden i ied in his s udy. Magni ica ions a e iden ical; he scale ba on he igh panel applies o all pic u es. (Spo es o he IPLA 35017 s ain we e no ob ained). J. Rod íguez e al. In e na ional Jou nal o Food Mic obiology 406 (2023) 110401 6 4. Discussion In his wo k, i e Epicoccum isola es belonging o h ee species we e eco e ed om a cheese ac o y. Epicoccum is a ubiqui ous genus ypi- cally ound in ai , soil, and decaying plan ma e ial (B aga e al., 2018). Al hough i can lead o an endophy ic li es yle (Chen e al., 2017), Epi- coccum has been ecen ly ecognized as a widesp ead plan pa hogen. In ag eemen wi h his, s ains o Epicoccum so ghinum ha e been epo ed o p oduce enuazonic acid (TeA), a po en phy o oxin blocking he pho osyn he ic pa hway (Oli ei a e al., 2019). Tenuazonic acid is conside ed unlikely o be o human heal h conce n (EFSA, 2011). Epi- coccum species can cause di e en ypes o spo s (colou , size, e c.) on he lea es (Be na di e al., 2022; Guo e al., 2020; Han e al., 2021; Liu e al., 2020; Mahade akuma e al., 2014; Niu e al., 2022; Xie e al., 2022) and o he o gans o plan s (Xiao e al., 2022; Xu e al., 2022). As epo ed in he li e a u e (B aga e al., 2018; Oli ei a e al., 2019), many Epicoccum species sec e e du ing g ow h pigmen s such as β- and γ-ca o ene, epicocconones, epipy ones, and o he s. Some o hese a e hough o be he cause o he eddish spo s on he p esen cheeses. S imula ion o ca o enoid p oduc ion in he yeas Xan hophyllomyces dend o hous by he ungus Epicoccum nig um has been epo ed (Echa- a i-E asun and Johnson, 2004). Howe e , in he p esen wo k, pigmen p oduc ion in he ilamen ous E. layuense IPLA 35011 was a ou ed when co-cul u ed wi h he yeas o m D. hansenii IPLA 34018. D. hansenii is among he yeas species wi h he highes p e alence on he su ace o ipened cheeses. An agonis ic and syne gis ic e ec s o he yeas on g ow h and enzyme ac i i y o se e al mould species ha e been epo ed (F ¨ ohlich-Wyde e al., 2019). Mos pigmen s and seconda y Epicoccum me aboli es (e.g., dike opipe azines, epico azines, epi- coccolides, epicocconig ones, epicocconones, epicolac one dime s, epi- py ones, la ipins, io nicins, e c.) show s ong an imic obial ac i i y agains bac e ia and ungi (Lee e al., 2020), o which p ope y ce ain Epicoccum s ains ha e been assessed as biocon ol agen s o c ops (B aga e al., 2018; Taguiam e al., 2021). O he seconda y me aboli es made by Epicoccum species (e.g., epicoccamides, me o e penoids, axol, e c.) ha e po en ac i i y agains cance cells (El-Sayed e al., 2020; Pe een e al., 2017), and could hus p o ide a sou ce o no el an i- cance d ugs. The isola ion o Epicoccum moulds om Spanish cheeses has ne e been epo ed. Indeed, li e a u e on he de ec ion o Epicoccum species in dai y p oduc s is sca ce. An Epicoccum pu pu ascens isola e was iden i- ied in Fon ina cheese as pa o a mic obial conso ium associa ed wi h a pink discolou a ion (Ca ini e al., 1979). Single s ains o E. nig um we e isola ed by Hocking and Faedo (1992) and Ku e and Skaa (2000) om Chedda (ou o 195 mould isola es) and No egia (ou o 159 mould isola es) cheeses, espec i ely. Two Epicoccum sp. isola es ha e been epo ed in he ungal communi ies o milk om cows wi h mas i is (Cos a e al., 1993). Fu he mo e, ew o no Epicoccum eads ha e been epo ed as componen s o he cheese mycobio a when examined using high h oughpu sequencing (HTS) me hods (Anelli e al., 2019; Biolca i e al., 2022). As in his s udy, signi ican a ia ions on he mac o- and mic o-mo phological ea u es o Epicoccum isola es om he same en i onmen ha e been al eady epo ed, wi h no associa ion o he phylogene ic esul s (Oli ei a e al., 2019). Ce ainly, Epicoccum is no conside ed a dai y ood spoilage agen (Cenci-Goga e al., 2021; Ga nie e al., 2017; Ku e e al., 2008). This genus is no included ei he in he lis o p io i y ungal pa hogens by he Wo ld Heal h O ganiza ion (WHO, 2022), and no epo o i s pa ho- genici y owa ds animals o humans was ound in da abases. Ne e - heless, E. nig um, a common species in ai and dus , can colonize he nasal issue o humans causing alle gic sinusi is (Noble e al., 1997), and indoo exposu e o his mould has been co ela ed wi h less as hma con ol (Segu a-Medina e al., 2019). The e o e, i any o hese symp- oms we e shown o inc ease among he wo ke s a he dai y plan , p o ec i e measu es in he ac o y should be aken. 5. Conclusions The ac ha a e y la ge numbe o cheeses in he a ec ed ac o y showed eddish-b own spo s sugges s Epicoccum had colonized he whole cheesemaking en i onmen . To elimina e such ex ensi e con amina ion, p olonged, pu pose ul ac ion will su ely be equi ed, pe haps including he ea men o equipmen and cheeses wi h highly ac i e, comme cial an i ungal agen s such as pima icin (na amycin). One o he s ains (E. layuense IPLA 35011) was eadily isola ed om a ain ed spo on cheese and could ep oduce he colou de ec on cheese slices, as did he wo E. i alicum s ains (IPLA 35013 and IPLA 35014). Genome analysis o hese s ains, and compa ison wi h Epicoccum iso- la es o di e en o igins, migh p o ide clues as o whe he adap a ions in hese bio ypes allowing coloniza ion o he dai y en i onmen ha e occu ed. 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