702

Journal of Food Protection, Vol. 76, No. 4, 2013, Pages 702–706 doi:10.4315/0362-028X.JFP-12-431

Research Note Distribution and Mycotoxigenic Potential of Section Nigri Species in Naturally Contaminated

JEFFREY D. PALUMBO* AND TERESA L. O’KEEFFE

U.S. Department of Agriculture, Agricultural Research Service, Plant Research Unit, Albany, California 94710, USA

MS 12-431: Received 28 September 2012/Accepted 9 December 2012

ABSTRACT In a previous study, inedible pick-out samples were assayed for aflatoxin and aflatoxigenic Aspergillus species. These samples contained high populations of black-spored Aspergillus section Nigri species. To investigate whether these species may contribute to the total potential mycotoxin content of almonds, Aspergillus section Nigri strains were isolated from these samples and assayed for ochratoxin A (OTA) and fumonisin B2 (FB2). The majority of isolates (117 strains, 68%) were identified as Aspergillus tubingensis, which do not produce either mycotoxin. Of the 47 and Aspergillus awamori isolates, 34 strains (72%) produced FB2 on CY20S agar, and representative strains produced lower but measurable amounts of FB2 on almond meal agar. No OTA-producing strains of Aspergillus section Nigri were detected. Almond pick-out samples contained no measurable FB2, suggesting that properly dried and stored almonds are not conducive for FB2 production by resident A. niger and A. awamori populations. However, 3 of 21 samples contained low levels (,1.5 ng/g) of OTA, indicating that sporadic OTA contamination may occur but may be caused by OTA-producing strains of other Aspergillus species.

The California almond industry produces 80% of the fumonisin B2 (FB2), one of several carcinogenic and world’s supply of almonds. After harvesting, almonds are cytotoxic fumonisin (14, 16). Fumonisins stored in stockpiles until they are transported to processors, currently are not regulated in commodities other than maize where they are hulled, shelled, sorted, graded, and either (11, 33), but the recent demonstration that FB2 produced by stored in bulk or packaged for sale. Sorting is performed at Aspergillus species can be found in grapes, wine, and raisins least once after shelling, particularly to remove nuts that (20–23, 35) suggests that these fungi might contribute to show visible mechanical or insect damage. Damaged nuts health risks in other commodities. are associated with increased risk for aflatoxin contamina- Although Aspergillus section Nigri species have been tion (31), and almonds that are sorted out (‘‘pick-outs’’) are found on almonds (5), very little is known about the risks of deemed inedible by processors. In the harvest years between mycotoxin contamination in this situation. Therefore, the 2001–2002 and 2010–2011, inedible nuts represented 1 to goals of this study were to characterize Aspergillus section 2% of each year’s crop (3). Nigri populations found in almond pick-out samples and to In a recent study of volatile compounds released from determine the mycotoxigenic potential of these populations. almond pick-out samples, Beck et al. (7) found high To accomplish these goals, both almond samples and aflatoxin levels and recovered Aspergillus flavus and isolated fungal strains were assayed for OTA and FB2 by Aspergillus parasiticus from these nuts. During that study, high-performance liquid chromatography (HPLC) methods. the authors also found a high incidence of black-spored MATERIALS AND METHODS aspergilli, members of Aspergillus section Nigri (6). This section contains several species, including A. carbonarius, Isolation of Aspergillus section Nigri strains. Twenty-one A. niger, A. awamori, A. tubingensis, and A. brasiliensis almond pick-out samples were provided previously by the Almond (1, 30, 34). A. carbonarius and certain strains of A. niger Board of California from almond processors in the California and A. awamori produce ochratoxin A (OTA), a mycotoxin Central Valley. As previously described (7), 7 of the 21 samples had been blanched during commercial processing (Table 1). characterized as nephrotoxic, carcinogenic, teratogenic, Almond samples were finely ground with an Electrolux Assistent immunosuppressive, and cytotoxic (12, 17, 18, 28). OTA food processor with a nut grater attachment (Magic Mill USA, concentrations in many commodities are regulated in the Upper Saddle River, NJ) and stored at 220uC until used. To European Union and other countries (10), which makes this enumerate total fungi and Aspergillus species from each sample, 3 g toxin an important consideration for exported tree nuts. In of ground almonds was added to 27 ml of sterile 0.05% Tween 80 addition, many strains of A. niger and A. awamori produce diluent and mixed by vortexing for 1 min. Almond suspensions were serially diluted in 0.05% Tween 80 diluent, and dilutions * Author for correspondence. Tel: 510-559-5876; Fax: 510-559-5737; were spread plated on dichloran–rose bengal–chloramphenicol E-mail: [email protected]. agar (DRBC). For recovery of A. carbonarius, dilutions were J. Food Prot., Vol. 76, No. 4 ASPERGILLUS SECTION NIGRI SPECIES IN ALMONDS 703

TABLE 1. Fungal populations recovered from almond samples Aspergillus section Nigri Mean ¡ SD total Sample no. population (CFU/g) Mean ¡ SD population (CFU/g) % of total fungal population

1 7.15 ¡ 0.07 | 104 2.40 ¡ 0.28 | 103 3.4 2 2.55 ¡ 0.64 | 104 1.05 ¡ 0.07 | 104 41.2 3a 6.65 ¡ 0.21 | 104 1.25 ¡ 0.50 | 104 18.8 4 1.80 ¡ 0.00 | 103 1.00 ¡ 0.00 | 103 55.6 5 7.00 ¡ 0.28 | 104 4.30 ¡ 0.42 | 104 61.4 6a 3.05 ¡ 0.21 | 104 3.00 ¡ 0.00 | 102 1.0 7a ,10 ,10 NDb 8 5.25 ¡ 1.91 | 103 3.70 ¡ 1.56 | 103 70.5 9 5.05 ¡ 1.77 | 104 4.60 ¡ 2.97 | 103 9.1 10a 9.00 ¡ 1.41 | 102 5.00 ¡ 0.00 | 102 55.6 11a 5.00 ¡ 7.07 | 101 5.00 ¡ 7.07 | 101 100 12 1.60 ¡ 0.00 | 104 3.40 ¡ 1.60 | 103 21.3 13 6.40 ¡ 0.71 | 103 3.30 ¡ 0.42 | 103 51.6 14a ,10 ,10 ND 15 2.00 ¡ 0.28 | 104 8.50 ¡ 0.42 | 103 42.5 16a 4.00 ¡ 2.83 | 102 ,10 ND 17 1.90 ¡ 0.28 | 105 6.15 ¡ 1.34 | 104 32.4 18 4.70 ¡ 0.14 | 104 2.55 ¡ 0.07 | 104 54.3 19 1.45 ¡ 0.37 | 104 6.50 ¡ 0.71 | 102 4.5 20 3.50 ¡ 1.13 | 104 1.00 ¡ 0.14 | 104 28.6 21 1.32 ¡ 0.11 | 105 1.20 ¡ 0.14 | 105 90.9 a Blanched almond samples. b ND, not determined.

plated on malt extract agar containing 10 mg/ml boscalid (MEA-B) Tool (BLAST) analyses (National Center for Biotechnology (29). Plates were incubated at 28uC for 3 days. Isolates of Information, Bethesda, MD). Aspergillus section Nigri, recognizable by dark brown to black sporulation, were transferred to potato dextrose agar for mainte- Mycotoxin analyses of almonds. For analysis of OTA and nance, and conidial suspensions of these isolates were stored in FB2 in each almond pick-out sample, 25 g of ground almonds and 0.05% Tween 80 plus 30% glycerol at 280uC. 2.5 g of NaCl were blended with 100 ml of 70% methanol for 1 min in a blender (Waring, Torrington, CT) and filtered through P8 Identification of Aspergillus species. DNA from Aspergillus coarse fluted filter paper (Fisher Scientific, Santa Clara, CA). strains was isolated as previously described (25). Conidia of each Lipids were partially removed by mixing 20 ml of filtrate with an fungal strain were inoculated into 1 ml of potato dextrose broth in equal volume of cyclohexane in a separatory funnel. After phase 1.5-ml microcentrifuge tubes and grown for 24 h at 28uC with separation, the aqueous fraction was collected and adjusted to 20 ml shaking at 150 rpm. Fungal biomass was collected by centrifugation with 70% methanol. For OTA purification, 10 ml of extract was at 15,000 | g for 10 min and homogenized with a Teflon diluted 1:5 with phosphate-buffered saline (PBS) plus 0.01% microcentrifuge tube pestle (Bel-Art Products, Pequannock, NJ) Tween 20 and filtered with 0.22-mm-pore-size Millex-GP poly- attached to a rotary tool (model 750, Dremel, Racine, WI). Genomic ethersulfone syringe filters (Millipore, Bedford, MA). Twenty DNA was isolated from homogenates with a MasterPure Yeast DNA milliliters of each diluted extract was applied to an OchraTest purification kit (Epicentre Biotechnologies, Madison, WI) and immunoaffinity column (VICAM, Milford, MA), columns were diluted 1:10 with sterile water for use as PCR templates. Fragments washed as recommended by the manufacturer, and OTA was of b-tubulin and calmodulin genes were amplified with primer eluted in 1.5 ml of methanol. To quantify OTA, 50 ml of each sets Bt2a (59-GGTAACCAAATCGGTGCTGCTTTC-39) and Bt2b sample was separated using an HPLC system (model 1100, Agilent (59-ACCCTCAGTGTAGTGACCCTTGGC-39) (15) and CF1L Technologies, Santa Clara, CA). Separations were performed on an (59-GCCGACTCTTTGACYGARGAR-39) and CF4 (59-TTTYTG- Inertsil ODS 3 column (5 mm, 4.6 by 250 mm; GL Sciences, Inc., CATCATRAGYTGGAC-39) (27), respectively. PCR conditions as Torrance, CA), with acetonitrile–0.1% phosphoric acid (65:35) as follows: 95uC for 5 min; 35 cycles of 95uC for 30 s, 61uC (for Bt2a/ the mobile phase at a flow rate of 1 ml/min. OTA was detected by Bt2b) or 52uC (for CF1L/CF4) for 30 s, and 72uC for 1 min; and fluorescence, with excitation and emission wavelengths of 333 and 72uC for 5 min. Amplified fragments were purified with the Clean & 460 nm, respectively. OTA was quantified from peak areas relative Concentrator-5 Kit (Zymo Research Corp., Irvine, CA), sequenced to a standard curve consisting of authentic OTA recovered from with the same primers with BigDye v. 3.1 cycle sequencing reagents ground almonds that were spiked with 0.25 to 10 ng/g authentic (Applied Biosystems, Foster City, CA), and run on a 3730 DNA OTA (Sigma-Aldrich Corp., St. Louis, MO). analyzer (Applied Biosystems). Sequences were aligned using For FB2 purification, 10 ml of extract (after partitioning with Lasergene sequence analysis software (DNASTAR, Madison, WI), cyclohexamide) was diluted 1:5 with PBS and filtered with 0.22- and strains were identified to species based on sequence homologies mm-pore-size Millex-GP polyethersulfone syringe filters. Twenty to reference strains as determined by Basic Local Alignment Search milliliters of each diluted extract was applied to a FumoniTest 704 PALUMBO AND O’KEEFFE J. Food Prot., Vol. 76, No. 4 immunoaffinity column (VICAM), columns were washed as recommended by the manufacturer, and fumonisins were eluted in 1.5 ml of methanol. Eluates were dried under a stream of nitrogen and redissolved in 600 mlof50% methanol. Eluates were analyzed for FB2 by HPLC after precolumn derivatization with o-phthaldialdehyde (OPA) (2). Derivatization reactions were performed using an Agilent model 1313A autosampler by mixing 30 ml of sample culture extracts and 120 ml of OPA reagent in the injector. After mixing (approximately 1 min), the entire derivatized sample (150 ml) was injected onto an Inertsil ODS 3 column (5 mm, 4.6 by 250 mm) and separated using a mobile phase of acetonitrile–water–acetic acid (67:32:1). Derivatized FB2 was detected by fluorescence, with excitation and emission wave- lengths of 335 and 440 nm, respectively. FB2 was quantified from peak areas relative to a standard curve constructed using 12.5 to 2,500 ng/ml authentic FB2 (Sigma-Aldrich Corp.) as standards.

Mycotoxin analyses of Aspergillus strains. To screen for OTA production, Aspergillus section Nigri strains were grown on FIGURE 1. FB2 production by A. niger (gray bars) and A. yeast extract–sucrose agar at 28 C for 7 days. Five agar plugs per u awamori (black bars) strains. Strains were screened for FB2 plate were removed with a number 3 cork borer (8 mm diameter) production after culture for 7 days on CY20S agar. and extracted with 1 ml of 100% methanol as described previously (8). Extracts were filtered with 0.2-mm-pore-size nylon syringe filters (Fisher Scientific) and analyzed for OTA by HPLC as Nigri isolates were identified by sequence analysis of b- described above. To screen for FB2 production by A. niger and A. tubulin and calmodulin gene fragments. Of these, 117 awamori isolates, strains were grown on Czapek yeast extract agar strains (68%) were identified as A. tubingensis and 47 with 20% sucrose (CY20S) (19) at 28uC for 7 days. Five agar strains (27%) were identified as A. niger (34 strains) or plugs per plate were extracted with 75% methanol as described A. awamori (13 strains). A. niger and A. awamori isolates (25). Extracts were filtered with 0.2-mm-pore-size nylon syringe were distinguished based on fixed sequence differences in filters and analyzed for FB2 by HPLC as described above. For calmodulin and b-tubulin genes between the two species, as quantification of FB production, A. niger and A. awamori strains 2 described by Perrone et al. (26). The remaining seven isolates were grown in triplicate on CY20S and almond meal agar (5% ground almonds plus 1.5% agar) for 7 days at 28uC. Agar plugs (4%)wereidentifiedasAspergillus foetidus or Aspergillus were extracted, and extracts were analyzed by HPLC as described piperis, which could not be resolved by b-tubulin and above. FB2 was quantified from peak areas as described above and calmodulin sequence comparisons. In contrast, in two separate normalized to agar plug weight. Differences in FB2 production by raisin vineyard studies, A. niger and A. awamori strains each strain on the two media were analyzed by a one-way analysis were recovered more frequently than were A. tubingensis of variance with Tukey’s post-test using GraphPad Instat (version strains (50 versus 45%, and 68 versus 25%,respectively)(25, 3.06, GraphPad Software, San Diego, CA). unpublished data).NoA. flavus or A. parasiticus strains were isolated from raisin samples, but unblanched almonds RESULTS AND DISCUSSION contained significant A. flavus and A. parasiticus populations Distribution of Aspergillus section Nigri species on (7). Together, these results suggest major differences in the almonds. Fungal propagules were readily recovered from fungal communities on these two crops. ground almond pick-out samples on DRBC plates (Table 1). In unblanched nuts, total fungal counts ranged from 103 to Mycotoxin production by Aspergillus isolates. None 105 CFU/g. Recovery of fungi from blanched nuts was more of the 171 Aspergillus section Nigri strains isolated from variable, ranging from ,10 CFU/g (samples 7 and 14) to almond pick-out samples produced detectable amounts of 104 CFU/g (samples 3 and 6). Colonies of Aspergillus OTA. This finding was not unexpected because A. section Nigri were recovered from all samples except carbonarius is the major OTA-producing species of the samples 7 and 14 at levels of 101 to 105 CFU/g. We did not black-spored aspergilli (1, 24), and no A. carbonarius observe any correlation between the relative frequencies of strains were isolated. Although some strains of A. niger and black-spored aspergilli and those of A. flavus and A. A. awamori produce OTA (26, 34), we did not isolate any parasiticus that were previously reported (7), especially OTA-producing strains of these species from almonds. We owing to the wide range of total fungal densities between detected no OTA production by A. tubingensis strains samples. No colonies appeared on MEA-B plates, indicating isolated from almonds, in agreement with recent reports that the absence of A. carbonarius in these almond samples. A. tubingensis is unlikely to produce OTA (24, 32). To examine the species diversity of Aspergillus section The 47 A. niger and A. awamori strains were screened Nigri recovered from almonds, we randomly picked 10 for FB2 production on CY20S, a high-osmolarity medium colonies (when possible) from plates of each almond sample that is conducive to FB2 production by these species (13). for further analysis. Fewer than 10 colonies were picked Of the 34 A. niger strains isolated from almonds, 25 strains from samples 6, 11, 16, and 19 because of poor recovery of (74%) produced FB2, and 9 (69%) of the 13 A. awamori black-spored aspergilli. A total of 171 Aspergillus section strains produced FB2. The frequency of FB2-producing J. Food Prot., Vol. 76, No. 4 ASPERGILLUS SECTION NIGRI SPECIES IN ALMONDS 705

and the ability of these strains to produce FB2 on almond meal agar suggests that almonds may be susceptible to FB2 contamination but only under conditions of high humidity or other improper storage conditions. Spurious OTA contamination was observed, but even in such low-quality almonds the OTA concentrations were quite low, indicating that OTA contamination of marketable almonds is likely to be much less common.

ACKNOWLEDGMENTS We thank Noreen Mahoney and Carlo Santillan for technical assistance and helpful discussions. This work was supported by the U.S. Department of Agriculture, Agricultural Research Service, Current Research Information System project 5325-42000-038-00.

FIGURE 2. Comparison of FB2 production by A. niger and A. awamori on CY20S and almond meal agar. Representative strains REFERENCES of A. niger and A. awamori were grown for 7 days on CY20S agar 1. Abarca, M. L., F. Accensi, J. Cano, and F. J. Caban˜es. 2004. (black bars) or 5% almond meal agar (gray bars) and assayed and significance of black aspergilli. Antonie Leeuwenhoek for FB2 production. Error bars represent standard deviations 86:33–49. of triplicate samples. Asterisks indicate strains that produced 2. Abbas, H. K., W. P. Williams, G. L. Windham, H. C. Pringle III, W. Xie, and W. T. Shier. 2002. Aflatoxin and fumonisin contamination significantly less FB2 on almond meal agar than on CY20S (P , 0.01). of commercial corn (Zea mays) hybrids in Mississippi. J. Agric. Food Chem. 50:5246–5254. 3. Almond Board of California. 2011. Almond almanac. Available at: strains is consistent with other studies, in which 66 to 77% http://www.almondboard.com/AboutTheAlmondBoard/Documents/ of strains of these species produced FB2 (22, 25, 35). The ALM110600_Almanac2011_LR.pdf. Accessed 24 September 2012. 4. Bayman, P., J. L. Baker, M. A. Doster, T. J. Michailides, and N. E. concentrations of FB2 produced were variable, ranging from 31.4 to 2,500 ng/ml (Fig. 1). The highest concentrations of Mahoney. 2002. Ochratoxin production by the Aspergillus ochraceus group and Aspergillus alliaceus. Appl. Environ. Microbiol. 68:2326– FB2 were produced by seven A. awamori strains. To further 2329. characterize the potential for FB2 production in almonds, A. 5. Bayman, P., J. L. Baker, and N. E. Mahoney. 2002. Aspergillus on niger and A. awamori strains producing high, moderate, and tree nuts: incidence and associations. Mycopathologia 155:161–169. low amounts of FB2 were grown on 5% almond meal agar 6. Beck, J., and N. Mahoney. Personal communication. as a surrogate substrate. In comparison to cultures grown on 7. Beck, J. J., N. E. Mahoney, D. Cook, and W. S. Gee. 2011. Volatile CY20S, all six strains produced less FB on almond meal analysis of ground almonds contaminated with naturally occurring 2 fungi. J. Agric. Food Chem. 59:6180–6187. agar, although the differences were significant (P , 0.01) in 8. Bragulat, M. R., M. L. Abarca, and F. J. Cabanes. 2001. An easy only three strains (A. niger strain APO20-5 and A. awamori screening method for fungi producing ochratoxin A in pure culture. strains APO13-7 and APO17-2) (Fig. 2). Int. J. Food Microbiol. 71:139–144. 9. Doster, M. A., and T. J. Michailides. 1994. Aspergillus molds and Ochratoxin and fumonisin content of almonds. aflatoxins in nuts in California. Phytopathology 84:583– 590. Despite the presence of FB2-producing aspergilli, no 10. European Commission. 2006. Commission Regulation (EC) No 1881/ detectable FB2 was found in the 21 almond pick-out 2006, setting maximum levels for certain contaminants in foodstuffs. samples. Three of the samples, however, contained Off. J. Eur. Union L 364:5–24. detectable amounts of OTA. OTA was recovered from 11. European Commission. 2007. Commission Regulation (EC) No 1126/ samples 10, 11, and 15 at 0.42, 1.21, and 0.59 ng/g, 2007, setting maximum levels for certain contaminants in foodstuffs respectively. The presence of OTA did not correlate with the as regards Fusarium toxins in maize and maize products. Off. J. Eur. Union L 255:14–17. concentration of aflatoxins detected in these same samples 12. Follmann, W., and S. Lucas. 2003. Effects of the mycotoxin (7). The low frequency and extent of OTA contamination ochratoxin A in a bacterial and a mammalian in vitro mutagenicity and the absence of detectable A. carbonarius populations test system. Arch. Toxicol. 77:298–304. suggest that in these samples OTA contamination was 13. Frisvad, J. C., J. Smedsgaard, R. A. Samson, T. O. Larsen, and U. caused by uncharacterized populations of other OTA- Thrane. 2007. Fumonisin B2 production by Aspergillus niger. J. Agric. producing Aspergillus species such as A. alliaceus, A. Food Chem. 55:9727–9732. 14. Gelderblom, W. C. A., N. P. J. Kriek, W. F. O. Marasas, and P. G. ochraceus, A. melleus, or other species in Aspergillus Thiel. 1991. Toxicity and carcinogenicity of the Fusarium monili- section Circumdati (4, 9). forme metabolite, fumonisin B1, in rats. Carcinogenesis 12:1247– Although the almond pick-out samples we used for 1251. study were of the lowest possible quality and unfit for 15. Glass, N. L., and G. C. Donaldson. 1995. Development of primer sets human consumption, our study reflects the potential for designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl. Environ. Microbiol. 61:1323–1330. almonds to be contaminated with Aspergillus species other 16. Gutleb, A. C., E. Morrison, and A. J. Murk. 2002. Cytotoxicity assays than aflatoxin-producing A. flavus and A. parasiticus. The for mycotoxins produced by Fusarium strains: a review. Environ. presence of FB2-producing A. niger and A. awamori strains Toxicol. Pharmacol. 11:309–320. 706 PALUMBO AND O’KEEFFE J. Food Prot., Vol. 76, No. 4

17. Haubeck, H. D., G. Lorkowski, E. Kolsch, and R. Roschenthaler. 27. Peterson, S. W. 2008. Phylogenetic analysis of Aspergillus species 1981. Immunosuppression by ochratoxin A and its prevention by using DNA sequences from four loci. Mycologia 100:205–226. phenylalanine. Appl. Environ. Microbiol. 41:1040–1042. 28. Petzinger, E., and K. Ziegler. 2000. Ochratoxin A from a 18. Kamp, H. G., G. Eisenbrand, J. Schlatter, K. Wurth, and C. toxicological perspective. J. Vet. Pharmacol. Ther. 23:91–98. Janzowski. 2005. Ochratoxin A: induction of (oxidative) DNA 29. Pollastro, S., R. M. D. M. Angelini, and F. Faretra. 2006. A new damage, cytotoxicity and apoptosis in mammalian cell lines and semi-selective medium for the ochratoxigenic fungus Aspergillus primary cells. Toxicology 206:413–425. carbonarius. J. Plant Pathol. 88:107–112. 19. Klich, M. A. 2002. Identification of common Aspergillus species. 30. Samson, R. A., P. Noonim, M. Meijer, J. Houbraken, J. C. Frisvad, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands. and J. Varga. 2007. Diagnostic tools to identify black aspergilli. Stud. 20. Knudsen, P. B., J. M. Mogensen, T. O. Larsen, and K. F. Nielsen. Mycol. 59:129–145. 2011. Occurrence of fumonisins B2 and B4 in retail raisins. J. Agric. 31. Schatzki, T. F., and M. S. Ong. 2001. Dependence of aflatoxin in Food Chem. 59:772–776. almonds on the type and amount of insect damage. J. Agric. Food 21. Logrieco, A., R. Ferracane, A. Visconti, and A. Ritieni. 2010. Natural Chem. 49:4513–4519. occurrence of fumonisin B in red wine from Italy. Food Addit. 2 32. Storari, M., L. Bigler, C. Gessler, and G. A. Broggini. 2012. Contam. 27:1136–1141. Assessment of the ochratoxin A production ability of Aspergillus 22. Mogensen, J. M., J. C. Frisvad, U. Thrane, and K. F. Nielsen. 2010. tubingensis. Food Addit. Contam. 29:1450–1454. Production of fumonisin B and B by Aspergillus niger on grapes 2 4 33. U.S. Food and Drug Administration, Center for Food Safety and and raisins. J. Agric. Food Chem. 58:954–958. Applied Nutrition, Center for Veterinary Medicine. 2001. Back- 23. Mogensen, J. M., T. O. Larsen, and K. F. Nielsen. 2010. Widespread ground paper in support of fumonisin levels in corn and corn products occurrence of the mycotoxin fumonisin B2 in wine. J. Agric. Food Chem. 58:4853–4857. intended for human consumption. U.S. Food and Drug Administra- 24. Nielsen, K. F., J. M. Mogensen, M. Johansen, T. O. Larsen, and J. C. tion, Center for Food Safety and Applied Nutrition, Center for Frisvad. 2009. Review of secondary metabolites and mycotoxins from Veterinary Medicine, College Park, MD. the Aspergillus niger group. Anal. Bioanal. Chem. 395:1225–1242. 34. Varga, J., J. C. Frisvad, S. Kocsube, B. Brankovics, B. Toth, G. 25. Palumbo, J. D., T. L. O’Keeffe, and J. A. McGarvey. 2011. Incidence Szigeti, and R. A. Samson. 2011. New and revisited species in

of fumonisin B2 production within Aspergillus section Nigri Aspergillus section Nigri. Stud. Mycol. 69:1–17. populations isolated from California raisins. J. Food Prot. 74:672–675. 35. Varga, J., S. Kocsube, K. Suri, G. Szigeti, A. Szekeres, M. Varga, B. 26. Perrone, G., G. Stea, F. Epifani, J. Varga, J. C. Frisvad, and R. A. Toth, and T. Bartok. 2010. Fumonisin contamination and fumonisin Samson. 2011. Aspergillus niger contains the cryptic phylogenetic producing black aspergilli in dried vine fruits of different origin. Int. species A. awamori. Fungal Biol. 115:1138–1150. J. Food Microbiol. 143:143–149.