Different grain grinding methods affect detection of Fusarium graminearum DNA and mycotoxins

167 Phy opa hologia Medi e anea (2017), 56, 1, 167−174 www. up ess.com/pm ISSN (p in ): 0031-9465 Fi enze Uni e si y P ess ISSN (online): 1593-2095 DOI: 10.14601/Phy opa hol_Medi e -20293 Co esponding au ho : T. Yli-Ma ila E-mail: [email p o ec ed] © 2017 Au ho (s). This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC-BY-4.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. TOOLS FOR FUSARIUM MYCOTOXIN REDUCTION IN FOOD AND FEED CHAINS RESEARCH PAPERS Di e en g ain g inding me hods a ec de ec ion o Fusa ium g aminea um DNA and myco oxins Tapani YLI-MATTILA1, Sa i RÄMÖ2, Taha HUSSIEN1,4, Ma ion RAUVOLA5, Veli HIETANIEMI3 and Jukka KAITARANTA5 1 Molecula Plan Biology, Depa men o Biochemis y, Uni e si y o Tu ku, FI-20014 Tu ku, Finland 2 Na u al Resou ces Ins i u e Finland (Luke), Managemen and P oduc ion o Renewable Resou ces, FI-31600 Jokioinen, Finland 3 Na u al Resou ces Ins i u e Finland (Luke), Bio-Based Business and Indus y, FI-31600 Jokioinen, Finland 4 Myco oxins Lab, Depa men o Toxins and Con aminan s, Na ional Resea ch Cen e , Cai o, Egyp 5 Tu ku Uni e si y o Applied Sciences, FI-20520 Tu ku, Finland Summa y. Fusa ium g aminea um is he mos impo an deoxyni alenol (DON) p oducing species in no he n Eu- ope and Asia. The highes DON le els in Finland ha e been ound in oa g ain, and DNA le els o F. g aminea um a e in ag eemen wi h DON le els, when DON is measu ed by acc edi ed GC-MS. In addi ion o DON, 3ADON is p esen in Finnish g ain samples. La ge a ia ions in DON con en and amoun s o F. g aminea um DNA, and poo coe icien o de e mina ion (R2) be ween hese, ha e been de ec ed in oa g ain when he RIDA®QUICK SCAN ki esul s o DON con en ha e been used. This s udy con i med ha he coe icien o de e mina ion was usually less when DNA o DON we e ex ac ed om oa lou , which was no g ound wi h 0.8 mm o 1 mm sie es. DON le els ob ained wi h he Rida®Quick me hod we e usually highe han hose ob ained wi h acc edi ed GC-MS in Finnish oa , ba ley and whea samples. The homogeniza ion o he oa lou by sie ing is he e o e likely o be connec ed o he a ia ions in DON de ec ion. Amoun s o F. g aminea um DNA and DON close o legisla i e limi s should be econ i med wi h acc edi ed quan i a i e analyses. Key wo ds: oa , ba ley, whea , myco oxins, qPCR. In oduc ion Fusa ium head bligh (FHB) was i s desc ibed in Eu ope in England in 1884 (Pa y e al., 1995). By 1924, FHB was al eady ound in England, Russia, Sweden, F ance, I aly, Ge many, Holland and No - way. Acco ding o Sundheim e al. (2013), he i s F. g aminea um isola e in No way was ound om oa in 1911, while in Finland he ungus was epo ed in 1932 (Rainio, 1932). A p esen , FHB is common in all Eu opean coun ies p oducing whea , maize, and o he ce eals such as ba ley and oa . Fusa ium g aminea um sensu s ic o, oge he wi h o he closely ela ed species o he F. g aminea um spe- cies complex, is he mos common cause o FHB in whea , ba ley, oa and o he small g ain ce eals in mos pa s o he wo ld. The main species o F. g aminea- um species complex in Eu ope is F. g aminea um sensu s ic o, which is also he mos impo an deoxyni ale- nol (DON) p oduce in mos coun ies in Eu ope (Pas- quali e al., 2016; Yli-Ma ila and Gagkae a, 2016). Fusa ium g aminea um DNA le els in oa a e as- socia ed wi h DON le els, when DON is measu ed by gas ch oma og aphy-mass spec ome y (GC- MS). When he Rida® Quick La e al Flow es esul s o DON con en we e compa ed o DNA le els o F. g aminea um, he ela ionships be ween he wo we e much less dis inc . The homogeniza ion o he oa lou du ing g inding o g ain and passage h ough a 1 mm sie e is likely o be connec ed o his a ia- ion (Yli-Ma ila e al., 2013). In he No dic coun ies, high le els o F. g aminea- um and DON ha e been ound especially in oa g ain, Phy opa hologia Medi e anea 168 T. Yli-Ma ila e al. while in no h-wes e n Russia F. g aminea um was ound only a e 2003 (Yli-Ma ila e al., 2009; Yli-Ma i- la and Gagkae a, 2010; F edlund e al., 2013; Sundheim e al., 2013). High g ain in es a ion le els o F. g aminea- um and amoun s o DON ha e caused se ious losses in ge mina ion a many ins ances e.g. in No way (Ki- men Seed Labo a o y, 2009). Inc eased F. g aminea um and DON le els may be associa ed wi h clima e change ( an de Fels-Kle x e al., 2012). These e ec s ha e led he equi emen o seed impo s in o No way. Recen ly, F. g aminea um has been sp eading no hwa d in Eu ope (e.g., Waalwijk e al., 2003; Ni- cholson e al. 2003; S epien e al., 2008; Nielsen e al., 2011; Talas e al., 2011; Somma e al., 2014; Sup oniene e al., 2010, 2016) displacing he closely ela ed F. cul- mo um. This shi may be due o changing ag icul- u al p ac ices, clima e change and inc eased maize c opping. In Finland, F. g aminea um was epo ed in ce eals in 1932 (Rainio, 1932) and in he 1960s (Uo i and Ylimäki, 1974), and his ungus has been p esen since hen (Ylimäki e al., 1979; Eskola e al., 2001). In no h-wes e n Russia F. g aminea um was ound mo e ecen ly (Yli-Ma ila e al., 2009). Fusa ium g aminea um is also p esen in No way, Sweden and Es onia (Yli-Ma ila e al., 2011). Acco ding o Sund- heim e al. (2013) and Aamo e al. (2015), F. g amine- a um has become he dominan DON-p oducing species in No way since 2000. Also in Finland, F. g aminea um is now he main DON-p oduce in ce e- als (Yli-Ma ila, 2010, Hie aniemi e al., 2016) The 3ADON chemo ype o F. g aminea um is dominan in mos no he n a eas, while he 15ADON chemo ype is p edomina ing in Cen al and sou h- e n Eu ope (Pasquali e al., 2016). The no h-wes e n Russian isola es belong o he 3ADON chemo ype, like Finnish isola es and unlike mos isola es om sou he n Russia. This sugges s ha he no h-wes - e n Russian F. g aminea um popula ions may ha e o igina ed om Finland, while he sou he n Russian popula ions may be mo e closely ela ed o he cen- al and sou he n Eu opean 15ADON popula ions, as sugges ed by Yli-Ma ila (2010). Se e al immunological me hods ha e been de el- oped o es ima ion o DON le els in ou ine analyses ca ied ou by ood labo a o ies. Howe e , an ibodies may show c oss- eac i i y wi h analogous compounds. Thus, DON can be o e es ima ed, wi h 15ADON, 3ADON, DON-3β-glucoside and o he DON de i a- i es, which a e masking compounds o med by plan de oxi ica ion ac i i y (Lemmens e al., 2005; Audenae e al., 2013) , while ma ix e ec s can lead o o e - o unde -es ima ion o myco oxins (Aamo e al., 2012). The aim o he p esen s udy was o in es iga e DON le els in Finnish ce eal g ain, ob ained wi h ac- c edi ed GC-MS and Rida®Quick me hods, o com- pa e hese wi h F. g aminea um DNA le els, and also measu e e ec s o g ain g inding me hods on DON and F. g aminea um DNA le els. Ma e ials and me hods Fusa ium isola e The single spo e isola e G243 o F. g aminea um was used as he s anda d in qPCR assays, as de- sc ibed by Yli-Ma ila e al. (2008, 2011). Myco oxin analyses Oa g ain samples (21 samples) o he yea 2013 we e a pa o he o al numbe o samples ana- lyzed o DON, 3ADON, 15ADON and o he i- cho hecenes a MTT Ag i ood Resea ch Finland (Na u al Resou ces Ins i u e Finland, FI-31600 Jo- kioinen, Finland). These we e selec ed om 125 oa samples, so ha in 19 samples had DON le els g ea - e han 1,750 ppb, one had 1,100 ppb DON and one had less han 200 ppb. In addi ion, he DON le els in 20 oa samples om he yea 2013 we e es ima ed us- ing Rida® Quick Scan myco oxin es s (R-Biopha m AG). These samples we e selec ed so ha 15 con- ained mo e han 1,750 pbb o DON and i e samples had less han 1,750 ppb. A MTT, he icho hecenes we e ex ac ed and analyzed wi h an acc edi ed GC- MS me hod, as desc ibed by Hie aniemi e al. (2004) and Yli-Ma ila e al. (2011). The g ain lou was p o- duced using a Falling numbe hamme mill KT-120 (Kone eollisuus Oy) using a 1 mm sie e in o de o ob ain homogenous samples and o achie e good epea abili y in myco oxin and DNA measu emen s. The g ain samples om a majo milling company we e used o Rida®Quick measu emen o DON and hey we e g ound using a sepa a e mill wi hou a sie ing p ocedu e p io o oxin measu emen . DNA ex ac ion A he Uni e si y o Tu ku, he 21 oa g ain sam- ples om MTT and he 20 samples om he milling company used o myco oxin analyses we e g ound 169 Vol. 56, No. 1, Ap il, 2017 G ain g inding and Fusa ium g aminea um de ec ion o DNA ex ac ion using a mill (K ups KM75 Co ee G inde ) wi hou sie ing as desc ibed by Yli-Ma ila e al. (2013). DNA was ex ac ed om g ound g ain samples and om he F. g aminea um isola e G243 s anda d using he GenElu e™ Plan Genomic DNA ki (Sigma-Ald ich), as desc ibed by Yli-Ma ila e al., (2008, 2009, 2013). Quan i a i e PCR The TMFg12 p ime s and p obe ha e been de- signed o he F. g aminea um speci ic RAPD-PCR p oduc (Yli-Ma ila e al., 2008). An IQTM5 Real- Time PCR De ec ion Sys em (Bio-Rad) was used o unning qPCR samples. Amoun s o Fusa ium spe- cies DNA in g ain samples we e de e mined as o al DNA quan i ied by a Qubi luo ome e (In i ogen), as desc ibed by Yli-Ma ila e al. (2011). Rida®Quick DON analyses Oa (30), whea (20) and ba ley (20) samples om di e en loca ions in Finland we e analyzed using he Rida®Quick DON me hod a Tu ku Uni e si y o Applied Sciences (TUAS), and he esul s we e com- pa ed wi h he acc edi ed GC-MS me hod desc ibed abo e. The ep oducibili y o he me hod was es ed wi h six pa allel samples om he h ee ce eals, wi h h ee di e en DON le els. G ound oa samples (12) we e also ob ained om an indus ial labo a o y (Labo a o y 1), and 12 g ound whea samples we e ob ained om a second indus ial labo a o y (Labo- a o y 2). G ain samples o compa ing he wo me h- ods o measu ing DON le els we e g ound using h ee mills. In Labo a o y 1 hey we e g ound using labo a o y mill KT 30 (Kone eollisuus OY) wi hou sie ing, while in he Labo a o y 2 he samples we e g ound by labo a o y mill 3100 Falling numbe wi h a sie e o 0.8 mm. A MTT, a labo a o y Falling num- be hamme mill wi h a 1 mm sie e was used as de- sc ibed abo e. Each g ound g ain sample (1 g) was placed in a Falcon ube and 15 ml o Rida®Quick DON ex ac- ion bu e was added. The mix u e was manually shaken o 3 min be o e cen i uga ion o 10 min (3,500 g). The clea supe na an (100 μL) was pipe - ed on o he applica ion a ea o he es s ip and he esul was ead a e 5 min. S a is ical analyses R2 (coe icien o de e mina ion), eg ession slope and P (signi icance o eg ession slope) we e calcu- la ed using he SigmaPlo e sion 12.0 (SPSS Inc.). The o iginal DNA and oxin concen a ions we e ans o med o loga i hmic alues [1 + lg (x)] o ob- ain no mal dis ibu ion o hese pa ame e s. Sam- ples gi ing concen a ions less han 0.5 mg kg-1 o g ea e han 5.5 mg kg-1 wi h Rida®Quick we e ex- cluded om he analyses, because hese alues we e ou side he linea pa o he de ec ion. The ep oducibili y be ween amoun s o DON es ima ed wi h Rida®Quick in oa g ain samples was es ed using h ee samples wi h DON le els o 1.4, 1.7 and 2.1 mg kg-1 (n = 6, Table 1). Comme cial s anda d (Check sample Deoxyni alenol in whea . Rome Labs. 1.431 ± 0.2566 mg kg-1) was also used. In ba ley, he DON le els we e 0.81, 1.30 and 1.60 mg kg-1, while in whea hey we e 0.79, 1.00 and 1.40 mg kg-1. Rida®Quick esul s we e compa ed o ac- c edi ed GC-MS esul s using eg ession analysis, and pai ed - es s we e applied o e eal he 95 % signi icance le el. Rela i e s anda d de ia ion (RSD %) was also calcula ed. Table 1. The ep oducibili y o DON de ec ion esul s (mean + SE o six DON ex ac ions and DON measu e- men s o each g ound g ain sample ob ained om MTT) in oa , ba ley and whea g ain samples wi h h ee di e en DON con en s. G ain MTT esul (mg kg-1) Rida®Quick esul (mg kg-1 ± SE) RSD % Oa 1.40 1.69 ± 0.04 5.27 1.70 1.98 ± 0.07 8.51 2.10 2.32 ± 0.09 9.87 Ba ley 0.81 1.80 ± 0.16 22.3 1.30 2.30 ± 0.12 12.7 1.60 2.32 ± 0.150 15.8 Whea 0.79 1.32 ± 0.06 22.6 1.00 1.59 ± 0.15 12.0 1.40 2.11 ± 0.10 10.3 Phy opa hologia Medi e anea 170 T. Yli-Ma ila e al. Resul s In he ep oducibili y analysis be ween he Rida®Quick and GC-MS de ec ion me hods, mos esul s ga e RSD pe cen ages equal o o less han 20, especially when he DON le els we e close o o abo e he allowed limi (Table 1). The co ela ion be ween F. g aminea um DNA and DON le els in Finnish oa samples om he yea 2013 was g ea e (R2 = 0.76), when he DNA was ex ac ed and measu ed om g ain samples g ound a MTT using a mill wi h a sie e. When he F. g aminea um DNA was measu ed om he same g ain samples g ound a he Uni e si y o Tu ku, using a me hod lacking he sie e, he co ela ion was less (R2 = 0.31, Figu e 1). The R2 alue om 20 oa samples om a ood company was 0.71, which was also less han o g ain samples g ound a MTT (Figu e 2). The e was a good co ela ion be ween he DON le els ob ained om 30 oa , 20 ba ley and 21 sp ing whea samples using acc edi ed GC-MS and he Rida®Quick me hod (Figu e 3). Pea son co ela- ion alues we e 0.935, 0.931 and 0.935, espec i ely, o oa , ba ley and sp ing whea samples. R2 alues we e 0.88, 0.80 and 0.89, espec i ely, o he oa , ba ley and sp ing whea samples. Howe e , pai ed - es s ( - alues 5.694, 7.854 and 3.544, espec i ely, o he h ee g ain ypes) showed di e ences a he 95% signi icance le el be ween he Rida®Quick and GC-MS esul s in all h ee ce eals. This was main- ly due o he g ea e DON alues ob ained using Rida®Quick me hod, including se en alse posi i e oa and i e alse posi i e ba ley and whea samples wi h Rida®Quick. 3ADON le els we e measu ed in oa samples and hey we e usually abou 10–20% o he DON le els. The ep oducibili y o DON le els is shown in Table 1. Comme cial whea s anda d (Rome Labs 1.431 mg kg-1) esul ed on a e age 2.11 mg kg-1 wi h RSD 21 %. When he DON le els om oa g ain samples g ound a MTT and analyzed wi h he acc edi ed GC-MS we e compa ed o hose ob ained om he same samples in wo indus ial labo a o ies and TUAS wi h he Rida®Quick me hod, he co ela ions we e high (R2 usually g ea e han 0.89; Figu e 4), al- hough DON amoun s ob ained wi h GC-MS we e usually less in oa , whea and ba ley samples (Table 1). The co ela ion was also good, when DON le els om whea g ain samples g ound in Labo a o y 2 wi h a mill ha ing a sie e we e compa ed wi h he Rida®Quick me hod in he h ee labo a o ies (R2 > Figu e 1. Co ela ion be ween log Fusa ium g aminea um DNA and log deoxyni alenol (DON) concen a ion in Finnish oa g ain samples in 2013. In all samples DON was measu ed a MTT using acc edi ed GC-MS. DNA was measu ed ei he om g ain samples g ound, a MTT, in a mill wi h a sie e o om he same g ain samples g ound wi h a mill wi hou a sie e, a UTU. Reg ession slopes and R2 (coe icien o de e mina ion) alues a e shown. * and *** indica e s a is ically signi ican (P<0.05 o 0.001). Figu e 2. Co ela ion be ween log Fusa ium g aminea um DNA and log deoxyni alenol (DON) concen a ions in Finnish oa g ain samples in 2013. DON le el was es i- ma ed using he RIDA® QUICK SCAN. Oa samples we e g ound by a milling company o DON analysis, o by Tu ku Uni e si y wi hou sie ing o DNA ex ac ion. Re- g ession slopes and R2 (coe icien o de e mina ion) alues a e shown. *** indica es s a is ically signi ican (P<0.001) R2 = 0.71*** R2 = 0.76*** R2 = 0.31* 171 Vol. 56, No. 1, Ap il, 2017 G ain g inding and Fusa ium g aminea um de ec ion Figu e 3. Co ela ions be ween log deoxyni alenol (DON) concen a ions in Finnish oa (a), ba ley (b) and whea (c) g ain samples in 2013, es ima ed using he RIDA® QUICK SCAN compa ed wi h o log DON concen a ions ob ained a MTT by using GC-MS. The samples we e g ound using he same mill a MTT o DON es ima ion, wi h sie ing. Reg ession slopes and R2 (coe icien o de e mina ion) alues a e shown. *** indica es s a is ically signi ican (P<0.001). Figu e 4. Co ela ions be ween log DON concen a ions in oa g ain p ocessed a wo milling company labo a o ies (Lab 1 and Lab 2), compa ed wi h DON concen a ions measu ed a TUAS and MTT. The g ain samples we e g ound a MTT (a) in a mill wi h a sie e, o a Lab 1 (b) in a mill wi hou a sie e. *** and ** indica e s a is ically signi ican (P<0.001 o 0.01). Phy opa hologia Medi e anea 172 T. Yli-Ma ila e al. 0.9, Figu e 5). In con as , when DON le els om oa g ain samples g ound in Labo a o y 1 wi h a mill wi hou a sie e we e compa ed wi h Rida®Quick me hod in he h ee labo a o ies, he co ela ions we e less co ela ed (usually < 0.8, Figu e 5). Discussion Ou esul s indica e ha he semiquan i a i e Rida®Quick me hod o DON de ec ion in mos cas- es me he equi emen s o he Commission Regula- ion (EC) 401/2006 o DON de e mina ion, since he RSD pe cen age alues we e equal o o less han 20. DNA le els o F. g aminea um in oa g ains we e, in all cases, in ag eemen wi h DON le els when DON was measu ed by acc edi ed GC-MS. When he Rida®Quick DON ki esul s we e compa ed o DNA le els o F. g aminea um, he a ia ion was much g ea e . Secu ing he homogeniza ion o he oa lou using a 1 mm sie e as compa ed o g inding wi hou a sie e was associa ed wi h he a ia ions o DON and F. g aminea um DNA le els. These esul s a e in ag eemen wi h p e ious esul s (Yli-Ma ila e al., 2013; Hie aniemi e al., 2016). Mo e alse posi i e esul s we e also ound in all h ee ce eals when using he Rida®Quick me hod as compa ed o acc edi ed GC-MS de ec ion. This was due o he gene ally highe DON le els ob ained wi h he Rida®Quick me hod. The alse posi i e esul s may ha e been a leas pa ially due o 3ADON ound de ec ed in oa samples. Excessi ely high alues o DON ha e also been p e iously de ec ed using some apid immunological es s (e.g. Aamo e al., 2012). The DON le els ob ained wi h he Rida®Quick me hod we e usually g ea e han hose ob ained Figu e 5. Co ela ions be ween log DON concen a ions in whea g ain samples g ound a Lab 2 (a) and MTT (b), bo h in mills wi h sie es. *** indica es s a is ically signi ican (P<0.001 o <0.01). 173 Vol. 56, No. 1, Ap il, 2017 G ain g inding and Fusa ium g aminea um de ec ion wi h he acc edi ed GC-MS me hod, in all h ee ce e- als. Howe e , he semiquan i a i e Rida®Quick DON me hod used he e me , in mos cases, he equi e- men s o he Commission egula ion (EC) 401/2006 o a quan i a i e DON de e mina ion. This should show RSD % equal o less han 20. Bu wi h low DON le els in ba ley and whea , he RSD % was abo e 20%. Fo consume s, low DON amoun s gua an ee ha he DON le els a e below he EU limi , bu o a me s hey mean ha hei c ops may be ejec ed due o alse posi i e DON alues. We conclude ha bo h RIDA® QUICK SCAN and qPCR can be ap- plied o sc eening o de ec ion o high DON le els in ce eal g ain samples, bu he Rida®Quick assays indica ing DON con en s close o he legisla i e lim- i s should be epea ed and, i necessa y, econ i med wi h an acc edi ed quan i a i e analysis. In u u e i is impo an ha assay esul s ob ained om di e - en apid immunoassay ki s used by ood indus ies a e compa ed o hose ob ained using acc edi ed chemical analyses. Acknowledgemen s This esea ch was inancially suppo ed by he Tu ku Uni e si y Founda ion, he Ol i Founda ion, he Egyp ian mission depa men , and a el g an s o PhD s uden Taha Hussien o he Uni e si y o Tu ku, om The Cen e o In e na ional Mobili y, Finland (CIMO). Li e a u e ci ed Aamo H.U., I.S. Ho gaa d, G. B odal, O., Elen, M. Jes oi and S.S. Klemsdal, 2012. 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