toxins Article Screening and Identification of Novel Ochratoxin A-Producing Fungi from Grapes Xiaoyun Zhang 1,†, Yulin Li 2,†, Haiying Wang 1, Xiangyu Gu 3, Xiangfeng Zheng 1, Yun Wang 1, Junwei Diao 1, Yaping Peng 1 and Hongyin Zhang 1,* 1 School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, China; [email protected] (X.Z.); [email protected] (H.W.); [email protected] (X.Z.); [email protected] (Y.W.); [email protected] (J.D.); [email protected] (Y.P.) 2 Hubei Key Laboratory, Edible Wild Plants Conservation and Utilization, 11 Cihu Road, Huangshi 435002, China; [email protected] 3 School of Grain Science and Technology, Jiangsu University of Science and Technology, 2 Mengxi Road, Zhenjiang 212003, China; [email protected] * Correspondence: [email protected]; Tel.: +86-511-8878-0174 † These authors contributed equally to this work. Academic Editor: Richard A. Manderville Received: 27 September 2016; Accepted: 8 November 2016; Published: 12 November 2016 Abstract: Ochratoxin A (OTA) contamination has been established as a world-wide problem. In this study, the strains with the ability of OTA production were screened by analyzing the green fluorescence of the isolates colonies from the grapes in Zhenjiang with 365 nm UV light and confirmed by HPLC with fluorescent detection (HPLC-FLD). The results showed that seven isolates acquired the characteristic of the fluorescence, of which only five showed the ability of OTA production as confirmed by HPLC-FLD analysis. The five OTA-producing strains were identified based on comparative sequence analysis of three conserved genes (ITS, BenA and RPB2) of the strains, and they are Talaromyces rugulosus (O1 and Q3), Penicillium commune (V5-1), Penicillium rubens (MQ-5) and Aspergillus aculeatus (MB1-1). There are two Penicillium species of the five OTA-producing strains and our study is the first to report that P. rubens, T. rugulosus and A. aculeatus can produce OTA. This work would contribute to comprehensively understanding the fungi with an OTA-producing ability in grapes before harvest and then take effective measures to prevent OTA production. Keywords: screening; identification; ochratoxin A (OTA); fungi; grapes 1. Introduction Many fungi produce mycotoxins that can cause acute or chronic intoxication and damage to humans and animals after ingestion of contaminated food and feed [1]. Among the mycotoxins, aflatoxins and ochratoxin A (OTA) occupy special places due to their high occurrence, widespread distribution and acute toxicity. As one of the first group of secondary metabolites of fungi that are toxic to animals, OTA, like aflatoxins started the distinctive science of mycotoxicology in the 1960s [2,3]. OTA is the most toxic member of the Ochratoxin group that includes OTA, its methyl ester, its ethyl ester (OTC), 4-hydroxyochratoxin A (4-OH OTA), ochratoxin B (OTB) and its methyl and ethyl esters and ochratoxin α (OTα). OTA is nephrotoxic, hepatotoxic, teratogenic and immunotoxic to animals and has been associated with fatal endemic human nephropathies [4,5]. Toxicity studies have revealed that OTA mainly affects the kidney and liver, after absorption from the gut and circulation via the portal vein, inducing hepatotoxicity in rats and hepatocellular carcinomas in mice [6]. The European Commission has established maximum allowable limits for this toxin in some food products. A tolerable daily intake (TDI) of no more than 5 ng OTA/kg bw/day is recommended [7]. Toxins 2016, 8, 333; doi:10.3390/toxins8110333 www.mdpi.com/journal/toxins Toxins 2016, 8, 333 2 of 14 The occurrence of OTA was mainly attributed to contamination by toxigenic molds with the ability of toxin production before or after harvesting. OTA is a ubiquitous mycotoxin produced by Aspergillus and Penicillium species that can be detected in a wide variety of agricultural commodities, livestock productsToxins 2016, 8, and333 processed food [7]. OTA was firstly described as a secondary2 metabolite of 14 of Aspergillus ochraceus from laboratory experiments [2]. In addition, there have been reports about the [6]. The European Commission has established maximum allowable limits for this toxin in some productionfood of OTAproducts. by otherA tolerable strains, daily mainly intake (TDI)Aspergillus of no more carbonarius than 5 ng OTA/kgand a bw/day small percentageis recommended of isolates of the closely[7]. related species Aspergillus niger [8–14]. Soon after the isolation of OTA from A. ochraceus, the formationThe of OTAoccurrence by Penicilliumof OTA was mainly viridicatum attributedwas to reportedcontamination [3]. by The toxigenic species molds was with later the correctly identified asabilityPenicillium of toxin production verrucosum before[15 or–17 after]. In harvesting. fact, the OTA natural is a ubiquitous occurrence mycotoxin and practical produced importance by of Aspergillus and Penicillium species that can be detected in a wide variety of agricultural commodities, OTA was firstlylivestock linked products to andPenicillium processedspecies food [7]. [OTA18,19 was]. Afterwards, firstly described some as a secondary other OTA-producing metabolite of strains of PenicilliumAspergillusgenus ochraceus were isolatedfrom laboratory and all experiments the OTA-producing [2]. In addition, strains there have were been separated reports about into the two large groups: P.production verrucosum of OTAand byPenicillium other strains, nordicum mainly Aspergillus[17,20 ].carbonarius In summary, and a smallA. ochraceuspercentage ofand isolatesP. verrucosum were the mainof the OTA closely producing related species species. Aspergillus However, niger [8–14]A. carbonarius. Soon after, as the another isolation major of OTA source from ofA. OTA, has ochraceus, the formation of OTA by Penicillium viridicatum was reported [3]. The species was later also been identifiedcorrectly identified mainly as in Penicillium grapes [verrucosum21]. [15–17]. In fact, the natural occurrence and practical Althoughimportance cereals of areOTA known was firstly to be linked particularly to Penicillium susceptible species to[18,19]. OTA Afterwards, contamination some [other22,23 ], there is an increasingOTA occurrence‐producing strains of OTA of Penicillium in a large genus variety were of isolated foods, and such all as the beer OTA [‐24producing,25] and strains milk were [26]. OTA has also been recentlyseparated reported into two large in grapes groups: and P. verrucosum grape juices and [ 27Penicillium–30]. The nordicum occurrence [17,20]. ofIn OTAsummary, in wine A. is linked ochraceus and P. verrucosum were the main OTA producing species. However, A. carbonarius, as to the presenceanother of major molds source on of grapes. OTA, has Previous also been studiesidentified have mainly shown in grapes that [21]. the occurrence of OTA is likely due to environmentalAlthough cereals conditions are known (climate) to be particularly and badly susceptible controlled to OTA harvesting contamination procedures, [22,23], there poor drying and inadequateis an increasing storage occurrence conditions of [OTA31,32 in]. a large variety of foods, such as beer [24,25] and milk [26]. The objectivesOTA has also of been this studyrecently were reported to screen in grapes the and natural grape juices OTA-producing [27–30]. The occurrence fungi related of OTA to in preharvest wine is linked to the presence of molds on grapes. Previous studies have shown that the occurrence decay andof OTA OTA contamination is likely due to of environmental grapes and conditions test their OTA(climate) production and badly capacitycontrolledin harvesting vitro with special attention toprocedures, those not poor reported drying and in literature.inadequate storage conditions [31,32]. The objectives of this study were to screen the natural OTA‐producing fungi related to 2. Resultspreharvest decay and OTA contamination of grapes and test their OTA production capacity in vitro with special attention to those not reported in literature. 2.1. Preliminary Screening of OTA-Producing Fungi by 365 nm Ultraviolet Light 2. Results One hundred and thirty-nine molds were isolated by macroscopic characters (colony growth 2.1. Preliminary Screening of OTA‐Producing Fungi by 365 nm Ultraviolet Light and colony morphology) and microscopic characters. The isolates were cultured for 7 or 14 days to accumulateOne metabolites hundred and in thirty the‐ solidnine molds culture were medium. isolated by Inmacroscopic 365 nm ultravioletcharacters (colony light, growth there was one and colony morphology) and microscopic characters. The isolates were cultured for 7 or 14 days to suspected accumulateAspergillus metabolitesisolate and in sixthe othersolid culture isolates medium. that produced In 365 nm green ultraviolet fluorescence light, there in was culture one medium. After fumigatingsuspected with Aspergillus 25%–28% isolate ammonia,and six other all isolates seven that of theproduced isolates green took fluorescence on enhanced in culture fluorescence intensity andmedium. were After recorded fumigating as positivewith 25%–28% strains. ammonia, The greenall seven fluorescence of the isolates of took O1, on one enhanced of the positive strains, influorescence 365 nm ultraviolet intensity and light were was recorded showed as positive in Figure strains.1a, The but green there fluorescence was no fluorescence of O1, one of the observed in positive strains, in 365 nm ultraviolet light was showed in Figure 1a, but there was no fluorescence the negativeobserved strain