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Journal of Food Protection, Vol. 74, No. 4, 2011, Pages 672–675 doi:10.4315/0362-028X.JFP-10-412

Research Note

Incidence of B2 Production within Aspergillus Section Nigri Populations Isolated from California Raisins

JEFFREY D. PALUMBO,* TERESA L. O’KEEFFE, AND JEFFERY A. MCGARVEY

Plant Research Unit, U.S. Department of Agriculture, Agricultural Research Service, 800 Buchanan Street, Albany, California 94710, USA

MS 10-412: Received 28 September 2010/Accepted 15 December 2010

ABSTRACT Fungi belonging to Aspergillus section Nigri occur frequently and in high populations on grapes. Species within this section include Aspergillus niger, A. tubingensis, and A. carbonarius, and they are potential sources for including A and fumonisin B2 (FB2) in grapes and grape products. Aspergillus section Nigri strains were isolated from California raisins to examine the frequency and extent of FB2 production. Of 392 strains isolated, 197 strains were identified as A. niger, 131 of which produced FB2. These strains produced from 1.2 to 27 mg/ml FB2 in culture. PCR amplification of fum1 and fum19 gene fragments showed that all FB2-producing strains and nearly all nonproducing strains of A. niger contain these genes. An additional 175 strains were identified as A. tubingensis, none of which produced FB2. PCR with fum1 and fum19 primers amplified gene fragments of 14 and 25% of A. tubingensis strains, respectively, suggesting that putative orthologs of A. niger fumonisin biosynthetic genes might occur in A. tubingensis. These results indicate that FB2 production is common among field isolates of A. niger and suggest that the potential for FB2 contamination of California raisins should be addressed further.

Grapes produced in California for use as table grapes, A. niger contains at least 10 genes orthologous to those wine grapes, and raisins are susceptible to bunch rot and in the 15-gene fumonisin biosynthetic cluster characterized postharvest decay diseases, most commonly caused by in F. verticillioides (28). Subsequent studies have demon- Aspergillus section Nigri spp. including A. niger and A. strated that the sequenced strain A. niger ATCC 1015 carbonarius (33). These fungi are common residents of (NRRL 328) produces FB2 but not FB1 in culture (9, 20). grape-growing regions worldwide, even in the absence of FB2 production has also been shown in A. niger strains disease. In addition, the major producer of the mycotoxin isolated from grapes, raisins, and other dried vine fruits (16, ochratoxin A in grapes has been shown to be A. carbonarius 22, 34). The detection of FB2 in wine (17, 23) strongly (6, 7, 15, 19, 29, 30). The most abundant species in grape implies the involvement of resident A. niger populations, and raisin vineyards, however, are species of the A. niger and suggests that other grape products, including raisins, ‘‘aggregate,’’ including A. niger, A. tubingensis, A. foetidus, could contain detectable levels of FB2 contamination. In and A. brasiliensis, so termed because they are morpholog- contrast to maize, suggested limits for in grape ically indistinguishable (1). products have not been established. Analysis of the sequenced genome of A. niger indicated The objectives of this study were to determine the the presence of a gene cluster orthologous to the fumonisin incidence of FB2-producing A. niger in California raisins and gene cluster in Fusarium verticillioides (teleomorph Gib- to examine Aspergillus section Nigri populations as a whole berella moniliformis) (5, 26). Fumonisins are polyketide for other fumonisin-producing species. In addition, PCR mycotoxins produced by F. verticillioides and several other amplification of fum1 and fum19 gene fragments was used to Fusarium spp. on maize and cereal crops (10, 21). The determine their utility in predicting fumonisin-producing major forms of fumonisin produced by Fusarium spp. are potential of Aspergillus strains. The fum1 gene encodes a B1 (FB1), B2 (FB2), and B3 (FB3), all of which have been putative polyketide synthase essential for fumonisin biosyn- shown carcinogenic and cytotoxic (11, 13). Accordingly, thesis, and fum19 encodes a putative ABC transporter shown total fumonisin levels in maize are suggested by the U.S. to be expressed under standard culture conditions (26). Food and Drug Administration not to exceed 2 to 4 ppm (32). In the European Union, regulatory limits for total MATERIALS AND METHODS fumonisins are 4 ppm for unprocessed maize and 0.2 to Fungal isolation from raisins. Aspergillus section Nigri 2 ppm for processed maize (3). strains were isolated from raisin vineyard samples. In brief, 10 raisin samples were taken from each of four vineyards located in Fresno County, CA, designated vineyards ‘‘F,’’ ‘‘B,’’ ‘‘K,’’ and * Author for correspondence. Tel: 510-559-5876; Fax: 510-559-5777; ‘‘M.’’ Each sample consisted of one to three dried-on-vine clusters. E-mail: [email protected]. From each sample, 10 raisins were placed onto each of three plates J. Food Prot., Vol. 74, No. 4 FUMONISIN B2–PRODUCING A. NIGER IN CALIFORNIA RAISINS 673 of dichloran–rose bengal–chloramphenicol agar containing 6% TABLE 1. Distribution of Aspergillus section Nigri spp. and NaCl and incubated at 28uC for 3 to 7 days. Ten colonies of black-, FB2-producing strains isolated from vineyard raisin samples yellow-, or green-spored Aspergillus were picked randomly from No. of Aspergillus isolatesa each raisin sample—100 isolates per vineyard, 400 isolates in all. Fungi were transferred to potato dextrose agar for maintenance and A. A. A. A. were stored at 270uC as conidial suspensions in 0.05% Tween 20, Vineyard niger tubingensis carbonarius uvarum Total 30% glycerol. F 53 (19) 40 (0) 0 (0) 3 (0) 96 (19) DNA isolation and PCR amplification of fumonisin B 52 (44) 45 (0) 0 (0) 2 (0) 99 (44) biosynthetic genes. Fungal strains were inoculated as conidia K 25 (21) 67 (0) 6 (0) 0 (0) 98 (21) into 1 ml of potato dextrose broth in 1.5-ml microcentrifuge tubes M 67 (47) 23 (0) 7 (0) 2 (0) 99 (47) and grown for 24 h at 28uC, with shaking. Fungal cells were Total 197 (131) 175 (0) 13 (0) 7 (0) 392 (131) collected by centrifugation at 15,000 | g for 10 min and a homogenized with a model 750 rotary tool (Dremel, Racine, WI) Numbers in parentheses represent number of FB2-producing fitted with a Teflon microcentrifuge tube pestle (Bel-Art Products, strains of each species. Pequannock, NJ). Genomic DNA was isolated from homogenates with the MasterPure Yeast DNA Purification Kit (Epicentre above demonstrated a recovery of 62% ¡ 3.3% of the total FB2 in Biotechnologies, Madison, WI). The fum1 primers, fum1.2f (59- the first extraction. Extractions with ultrasonication, described CCATCGTGGGATCTCAGAGATG-39) and fum1.2r (59-CGCC- elsewhere (9), showed equivalent recovery rates. Thus, total FB2 AATGTCAAGCATATGGTC-39), and fum19 primers, fum19.2f production was corrected for recovery efficiency by dividing (59-CCACGCTGTTGGGACTGAACTA-39) and fum19.2r (59- calculated FB2 values by 0.62. GCGTTGCGAAGTGTTCAATAGC-39), were designed to gener- ate 557- and 536-bp products, respectively, by using as templates RESULTS AND DISCUSSION the corresponding gene sequences from the genome sequence of A. niger strain ATCC 1015 (4). Amplification conditions were 95uC Incidence of Aspergillus section Nigri spp. and of for 5 min; 35 cycles of 95uC for 30 s, 57uC for 30 s, 72uC for 1 min; FB2-producing strains. Over 97% of the Aspergillus and 72uC for 5 min. For species identification of each fungal strains isolated from raisins belonged to section Nigri; isolate, primers Bt2a (59-GGTAACCAAATCGGTGCTGCTTTC- strains of other Aspergillus spp. were chosen for isolation as 39) and Bt2b (59-ACCCTCAGTGTAGTGACCCTTGGC-39) (12) part of a parallel study of ochratoxin A production (25). The were used to amplify b-tubulin gene fragments, by using the same majority of black Aspergillus isolates were identified as A. amplification conditions as above, except the primer annealing niger or A. tubingensis, accounting for 91 to 98% of the temperature was 61uC. Amplified b-tubulin gene fragments were isolates collected from each vineyard (Table 1). A. carbo- sequenced with the Bt2a primer, and DNA sequence homologies narius strains represented less than 4% of the collection, and to known Aspergillus spp. were determined by Basic Local Alignment Search Tool (BLAST) analysis (National Center for A. uvarum strains represented less than 2%. A. uvarum is a Biotechnology Information, Bethesda, MD). uniseriate species of black Aspergillus (27), implicated as one of several species involved in Aspergillus rot of grape in

FB2 analysis. Fungal isolates were grown on Czapek yeast Europe, particularly in years with late-season rain prior to extract agar with 20% sucrose (CY20S) (14) for 4 days at 28uC. harvest (18). The distribution of A. niger and A. tubingensis Five agar plugs per plate were cut with a no. 3 cork borer (8-mm varied among vineyards sampled. Isolates from vineyards F diameter) and extracted with 1 ml of 75% methanol at room and B were divided nearly evenly between the two species, temperature for 2 h. Extracts were filtered with 0.2-mm nylon but A. tubingensis was isolated nearly three times more syringe filters (Fisher Scientific, Santa Clara, CA) and analyzed for frequently from vineyard K, and A. niger was isolated nearly FB2 by high-performance liquid chromatography (HPLC) after three times more frequently from vineyard M. This uneven precolumn derivatization with o-phthaldialdehyde (OPA) (2, 8). distribution in the latter two vineyards may be indicative of OPA reagent was prepared by dissolving 60 mg of OPA in 1.5 ml of methanol, diluting it with 7.5 ml of 0.1 M sodium tetraborate, environment-specific ecological interactions between these and adding 75 ml of 2-mercaptoethanol. OPA reagent was species, and may be reflective of more complex interactions dispensed into 2-ml autosampler vials for automated derivatization among coexisting Aspergillus section Nigri populations. reactions on a model 1100 HPLC system with a model 1313A Further sampling with statistical models is necessary to autosampler (Agilent Technologies, Santa Clara, CA). Sample describe the geographic distribution of these fungi. culture extracts (30 ml) and OPA reagent (120 ml) were mixed Production of FB2 was observed in 66% of A. niger together in the injector and held for 10 min for derivatization strains (Table 1). Within samples from each vineyard, FB2- reaction to occur. The entire derivatized sample (150 ml) was producing strains ranged from 36 to 85% of the A. niger injected onto an Inertsil ODS-3 5-mm column (4.6 by 250 mm; isolates, and from 20 to 48% of the total collection of black Varian, Inc., Palo Alto, CA), and separated with a mobile phase of Aspergillus isolates. No strains of A. tubingensis, A. acetonitrile–water–acetic acid (75:25:1) at a flow rate of 1 ml/min. carbonarius, or A. uvarum produced FB . This is consistent Derivatized FB was detected with an Agilent model 1321A 2 2 with other studies in which strains of these species, isolated fluorescence detector, with excitation and emission wavelengths of from grapes (16) or raisins (22), did not produce detectable 335 and 440 nm, respectively. FB2 in each sample was quantified relative to a standard curve constructed with 10 to 1,000 ng of amounts of FB2. authentic FB2 (Sigma-Aldrich Corp., St. Louis, MO) per injection. Among FB2-producing strains, we observed a large Repeated extraction of agar plugs from triplicate cultures of two variability in the amount of FB2 produced on CY20S agar different FB2-producing strains by using the protocol described (Fig. 1). Vineyard F isolates produced 1.3 to 12 mg/ml, 674 PALUMBO ET AL. J. Food Prot., Vol. 74, No. 4

TABLE 2. Correlation of FB2 production and PCR amplification of fum1 and fum19 genes in Aspergillus nigera

FB2 fum1 PCR fum19 PCR Vineyard production product product

F19z 19z 19z 342 34z 32z B44z 44z 44z 82 5z 7z K21z 21z 21z 42 3z 3z M47z 47z 47z 202 20z 20z Total 131z 131z 131z 662 62z 62z

a Numbers indicate number of A. niger isolates analyzed.

Further experimentation is necessary to determine the presence and expression of each gene involved in fumonisin biosynthesis, in both FB2-producing and nonproducing strains. This will demonstrate the genetic and metabolic FIGURE 1. Production of FB2 by Aspergillus section Nigri strains in culture media. Isolates from vineyard F (dark gray diversity affecting fumonisin production within A. niger bars), vineyard B (white bars), vineyard K (light gray bars), and populations. In contrast to A. niger, in 175 A. tubingensis vineyard M (hatched bars) were grown on CY20S agar and strains, 24 strains yielded fum1 and 43 strains yielded fum19 analyzed for FB2 by HPLC. FB2 levels were quantified relative to a PCR products. This could indicate either the presence of standard curve and corrected for extraction efficiency. orthologous genes within a partial or nonfunctional fumonisin biosynthetic gene cluster, or amplification of vineyard B isolates produced 1.5 to 14 mg/ml, vineyard K fragments of similar but unrelated genes. DNA hybridiza- isolates produced 1.9 to 20 mg/ml, and vineyard M isolates tion analyses to detect each target gene in A. tubingensis will produced 1.2 to 27 mg/ml. This was in agreement with the be useful in identifying potential orthologous genes and the range of FB2 production by A. niger strains in other studies. extent of homology to A. niger. Mogensen et al. (24) observed a range of 2.9 to 25 mg/ml This report demonstrates that California raisins might FB2 produced by five strains of A. niger on Czapek yeast harbor diverse populations of Aspergillus section Nigri, and extract agar with 5% NaCl. Twenty strains of A. niger and within those populations, there exists a strong potential for FB2 A. awamori assayed by Varga et al. (34) produced from production. Therefore, FB2 contamination of raisins should be 0.017 to 19.6 mg/g FB2 on CY20S agar. In those studies, of concern to producers and consumers. Examination of FB2 FB2 was measured with liquid chromatography–mass levels in California raisins, throughout all stages of processing, spectrometry methods, which allowed for both quantitation is necessary for risk assessment and to develop mitigation and compound identification. Identification of FB2 by using approaches to minimize mycotoxin exposure. In addition, OPA derivatization, in contrast, is based on comparison of ecological studies of fumonisin-producing and -nonproducing retention time with authentic FB2. From the present study, it Aspergillus spp. and strains may lead to strategies for is not clear whether FB2 production or the amount of FB2 biological control of fumonisin contamination of the crop. produced confers an ecological fitness advantage to individual strains of A. niger or to Aspergillus populations ACKNOWLEDGMENTS as a whole. We thank N. Mahoney for technical advice and S. Vasquez for raisin samples. This work was supported by U.S. Department of Agriculture, Detection of fum1 and fum19 genes by PCR and Agricultural Research Service, Cooperative Research Information Service project 5325-42000-036-00D. correlation with FB2 production. All FB2-producing isolates of A. niger yielded PCR products when genomic REFERENCES DNA was amplified with fum1 and fum19 primers 1. Abarca, M. L., F. Accensi, J. Cano, and F. J. Cabanes. 2004. (Table 2). In addition, 94% of the FB2-nonproducing A. Taxonomy and significance of black aspergilli. Antonie Leeuwenhoek niger isolates yielded one or both PCR products. This 86:33–49. suggests that the presence of these genes in A. niger is not 2. Abbas, H. K., W. P. Williams, G. L. Windham, H. C. Pringle III, W. predictive of FB2 production. In contrast, Susca et al. (31) Xie, and W. T. Shier. 2002. and fumonisin contamination detected the presence of fum8 in 9 of 9 FB2-producing A. of commercial corn (Zea mays) hybrids in Mississippi. J. Agric. Food niger strains, but in only 2 of 28 nonproducing strains. Chem. 50:5246–5254. 3. Anonymous. 2007. Commission Regulation (EC) No 1126/2007, Taken together, these data indicate that in nonproducing setting maximum levels for certain contaminants in foodstuffs as strains of A. niger, some or all of the genes in the fumonisin regards Fusarium in maize and maize products. Off. J. Eur. biosynthetic gene cluster may be present but not functional. Union L 255:14–17. J. Food Prot., Vol. 74, No. 4 FUMONISIN B2–PRODUCING A. NIGER IN CALIFORNIA RAISINS 675

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