toxins Article Improvement of the Culture Medium for the Dichlorvos-Ammonia (DV-AM) Method to Selectively Detect Aflatoxigenic Fungi in Soil Kimiko Yabe 1,*, Haruna Ozaki 1, Takuya Maruyama 1, Keisuke Hayashi 1, Yuki Matto 1, Marika Ishizaka 1, Takeru Makita 1, Syun-ya Noma 1, Kousuke Fujiwara 1 and Masayo Kushiro 2 1 Department of Environmental and Food Sciences, Faculty of Environmental and Information Sciences, Fukui University of Technology, 3-6-1 Gakuen, Fukui-shi, Fukui 910-8505, Japan; [email protected] (H.O.); [email protected] (T.M.); [email protected] (K.H.); [email protected] (Y.M.); [email protected] (M.I.); [email protected] (T.M.); [email protected] (S.-y.N.); [email protected] (K.F.) 2 Food Research Institute, National Agriculture and Food Research Organization (NARO), 2-1-12 Kannon-dai, Tsukuba-shi, Ibaraki 305-8642, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-776-292-408 Received: 10 November 2018; Accepted: 30 November 2018; Published: 5 December 2018 Abstract: The dichlorvos-ammonia (DV-AM) method is a simple but sensitive visual method for detecting aflatoxigenic fungi. Here we sought to develop a selective medium that is appropriate for the growth of aflatoxigenic fungi among soil mycoflora. We examined the effects of different concentrations of carbon sources (sucrose and glucose) and detergents (deoxycholate (DOC), Triton X-100, and Tween 80) on microorganisms in soils, using agar medium supplemented with chloramphenicol. The results demonstrated that 5–10% sucrose concentrations and 0.1–0.15% DOC concentrations were appropriate for the selective detection of aflatoxigenic fungi in soil. We also identified the optimal constituents of the medium on which the normal rapid growth of Rhizopus sp. was completely inhibited. By using the new medium along with the DV-AM method, we succeeded in the isolation of aflatoxigenic fungi from non-agricultural fields in Fukui city, Japan. The fungi were identified as Aspergillus nomius based on their calmodulin gene sequences. These results indicate that the new medium will be useful in practice for the detection of aflatoxigenic fungi in soil samples including those from non-agricultural environments. Keywords: aflatoxins; Aspergillus nomius; Aspergillus parasiticus; culture medium; dichlorvos- ammonia (DV-AM) method; deoxycholate; detergent; Rhizopus sp. Key Contribution: A selective medium that is appropriate for the growth of aflatoxigenic fungi in soil was devised: and we could successfully detect aflatoxigenic fungi isolated from non-agricultural fields in Fukui city, Japan. 1. Introduction Aflatoxins (AFs) are highly toxic, carcinogenic, and teratogenic secondary metabolites produced by fungi [1]. The contamination of agricultural commodities with AFs is not only a serious health hazard to humans and animals, causing hepatotoxicity as an acute toxicity or hepatic cancer as a chronic toxicity [2] but also a cause of huge economic losses worldwide. AFs are produced mainly by certain strains of Aspergillus flavus Link and Aspergillus parasiticus Speare [1]. These fungi have been thought to be distributed mostly in tropical and subtropical Toxins 2018, 10, 519; doi:10.3390/toxins10120519 www.mdpi.com/journal/toxins Toxins 2018, 10, 519 2 of 14 regions [3–5]. Aflatoxigenic fungi are present in soil as conidia, sclerotia, or hyphae, which act as primary inoculants for directly infecting peanuts or for infecting aerial crops such as maize, sugarcane, and cotton [6]. These fungi have also been reported in temperate regions (especially when droughts have occurred) in the United States and other countries [3]. Several other species of the Aspergillus group have been reported to produce AFs: A. nomius [7–9], A. pseudotamarii [10,11], A. bombycis [12], A. parvisclerotigenus [13–15], A. minisclerotigenes [13], and more, most of which are reported to occur in temperate regions. Additional unknown aflatoxigenic species mayToxins be present, 2018, 7, x FOR and PEER they REVIEW may be widely distributed in the world. The clarification of the distribution2 of 14 as well as the habitats of aflatoxigenic fungi is important to effectively prevent aflatoxin contamination in crops,[3–5]. feed,Aflatoxigenic and food. fungi are present in soil as conidia, sclerotia, or hyphae, which act as primary inoculantsMany researchers for directly have infecting reported peanuts the isolationor for infe ofcting aflatoxigenic aerial crops fungi such fromas maize, the soil sugarcane, in agricultural and cotton [6]. These fungi have also been reported in temperate regions (especially when droughts have fields (e.g., cotton, peanut and maize fields), and the researchers usually used multiple procedures: occurred) in the United States and other countries [3]. (1) the isolation of Aspergillus section Flavi using Aspergillus flavus and parasiticus agar (AFPA) Several other species of the Aspergillus group have been reported to produce AFs: A. nomius [7– medium [16], modified rose Bengal agar (MRBA) medium [17], or coconut agar medium [18]; (2) the 9], A. pseudotamarii [10,11], A. bombycis [12], A. parvisclerotigenus [13–15], A. minisclerotigenes [13], and confirmationmore, most of of aflatoxin which are production reported to by occur the isolatedin temperate strains regions. with theAddition use ofal thin unknown layer chromatography aflatoxigenic (TLC),species high-performance may be present, liquid and they chromatography may be widely (HPLC) distributed [19– 22in], the an world. enzyme-linked The clarification immunosorbent of the assaydistribution (ELISA) as [23 well–28 ],as andthe habitats other methods;of aflatoxigeni (3)c thefungi identification is important to of effectively aflatoxigenic prevent fungi aflatoxin using a polymerasecontamination chain in reaction crops, feed, (PCR) and orfood. a real-time PCR [29–31], and other methods. Since methods (1) and (3)Many are researchers useful for discriminatinghave reported the fungi isolation belonging of aflatoxigenic to Aspergillus fungisection from the Flavi, soil in the agricultural ability of the fungifields to produce (e.g., cotton, aflatoxins peanut needand maize to be confirmedfields), and bythe anotherresearchers analytical usually methodused multiple noted procedures: in method (2). These(1) methodsthe isolation are usefulof Aspergillus and fruitful section techniques Flavi using but Aspergillus are time-consuming flavus and parasiticus and often agar require (AFPA) some specialmedium facilities, [16], modified especially rose in Bengal step (2). agar For (MRBA) more practical medium use,[17], aor single-step coconut agar method medium for [18]; the (2) selective the detectionconfirmation of aflatoxigenic of aflatoxin fungi production from environmental by the isolated strains samples with is the desirable. use of thin layer chromatography (TLC),We previously high-performance developed liquid a sensitive chromatography and simple (HPLC) visual [19–22], detection an enzyme-linked method, the dichlorvos-ammonia immunosorbent (DV-AM)assay (ELISA) method [[23–28],32], in whichand other fungi methods; are first cultured(3) the identification on AF-inducible of aflatoxigenic agar medium fungi supplemented using a polymerase chain reaction (PCR) or a real-time PCR [29–31], and other methods. Since methods (1) with dichlorvos (DV) for 2–6 days, and the resulting colonies are then treated by ammonia (AM) vapor and (3) are useful for discriminating fungi belonging to Aspergillus section Flavi, the ability of the to monitor the mycelial color changes from pale yellow to brilliant purple-red. The color change of the fungi to produce aflatoxins need to be confirmed by another analytical method noted in method (2). colonyThese is duemethods to color are changesuseful and of fruitful aflatoxin techniques precursors, but i.e.,are time-consuming versiconal hemiacetal and often acetate require (VHA) some and versiconolspecial facilities, acetate (VOAc), especially which in step accumulate (2). For more in practical the mycelia use, bya single-step the inhibition method of an for AF the biosynthetic selective enzyme,detection VHA of esterase,aflatoxigenic by DVfungi [33 from] (Figure environmental1A). samples is desirable. FigureFigure 1. Outline 1. Outline of of the the DV-AM DV-AM method. method.DV DV inhibits the the versiconal versiconal hemiacetal hemiacetal acetate acetate (VHA) (VHA) esterase esterase in AFin AF biosynthesis biosynthesis to to cause cause an an accumulation accumulation of of VH VHAA and and versiconol versiconol acetate acetate (VOAc) (VOAc) instead instead of AFs of in AFs in thethe mycelia.mycelia. AM AM vapor vapor treatment treatment change change the colors the colorsof these of substances these substances from pale fromyellow pale to brilliant yellow to brilliantpurple-red. purple-red. Mutation Mutation sites of A. sites parasiticus of A. parasiticus NIAH-26 andNIAH-26 95DM strains and 95DM are also strains indicated arealso (gray indicated thick (grayarrows). thick arrows). AFs: aflatoxins, AFs: aflatoxins, AM: ammonium AM: ammonium vaper, NA: vaper, norsolorinic NA: norsolorinic acid, VHOH: acid, versiconal, VHOH: versiconal, VOH: VOH:versiconol. versiconol. SinceWe these previously precursors developed contain a sensitive anthraquinone and simple moieties visual in detection their structures, method, the the mycelial dichlorvos- colors areammonia markedly (DV-AM) changed method from the [32], original in which yellowish fungi are colors first