Impact of fungicides on Aspergillus carbonarius growth and ochratoxin A production on synthetic grape-like medium and on grapes

A study was undertaken to evaluate the impact of the application of several fungicide treatments used in Spanish vines on Aspergillus carbonarius growth and ochratoxin A production. Three trials were designed in order: (1) to screen 26 fungicides at the doses recommended by manufacturers on grape-like synthetic medium at 20 and 30°C; (2) to find out the minimum inhibitory concentration of each fungicide for A. carbonarius growth on synthetic medium; and (3) to investigate the effect of several fungicides on A. carbonarius-inoculated grapes. In synthetic medium nine fungicides significantly reduced A. carbonarius growth rate. Meanwhile, 13 fungicides completely inhibited its growth. In general, growth was faster at 30°C than at 20°C, contrary to ochratoxin A production. Fungicides that stopped fungal growth also inhibited ochratoxin A production, but not all the fungicides that reduced growth reduced the ochratoxin A synthesis. In general, fungicides that contained copper or strobilurins reduced both growth and ochratoxin A production, contrary to sulphur fungicides. At the optimum temperature for A. carbonarius growth of 30°C, higher amounts of fungicide were needed to prevent fungal growth than at 20°C. Among the fungicides that inhibited A. carbonarius growth on synthetic medium at the initial doses, cyprodinil seemed to be the active ingredient more effective at stopping fungal growth when testing reduced doses. The fungicide effect on grapes was similar to that on synthetic medium. Both infection and ochratoxin A production were reduced when using cyprodinil (37.5%) plus fludioxonil (25%) and azoxystrobin (25%). Penconazole (10%) also showed a clear reduction in ochratoxin A production at both temperatures, although infection was only reduced at 20°C. Ochratoxin A reduction was strain and temperature-dependent. In general, fenhexamid (50%), mancozeb (80%) and copper hydroxide (80%) plus copper (50%) enhanced infection and ochratoxin A production.     

Occurrence of ochratoxin A and toxigenic potential of fungal isolates from Spanish grapes

This paper reports the results from an extensive survey of filamentous fungi isolated from wine‐producing grapes and their capacity to produce ochratoxin A (OTA) on Czapek Yeast Autolysate agar (CYA), in order to assess their potential for producing this toxin in grapes. Grapes were sampled from four Spanish wine‐producing regions during 2001. The fungal infection in berries increased with time, reaching 100% at harvest. A total of 386 isolates of Aspergillus section Nigri and 10 of Aspergillus section Circumdati were isolated and tested for their ability to produce OTA in CYA. 21 strains produced OTA (18 Aspergillus section Nigri and 3 Aspergillus section Circumdati). Aspergillus section Circumdati isolates produced higher amounts of OTA than Aspergillus section Nigri ones, with means of 10.76 µg g^?1 CYA and 1.42 µg g^?1 CYA, respectively. Despite this, black aspergilli are believed to be highly responsible for the OTA levels found in musts and wines, as it is more widespread in grapes. Musts (n = 40) produced from the grapes collected were analysed. 15% were found to contain OTA, concentrations ranging from 0.091 to 0.813 ng ml^?1 (detection limit: 0.07 ng ml^?1), but no correlation was found with the ochratoxigenic moulds isolated from grapes. Copyright © 2004 Society of Chemical Industry     

Kinetics of Ochratoxin A Production and Accumulation by Aspergillus carbonarius on Synthetic Grape Medium at Different Temperature Levels

ABSTRACT:  The effects of incubation time (up to 10 d) and temperature (7, 15, 20, 25, 30, 35, and 42 °C) on mean ochratoxin A (OTA)-producing capacity and OTA accumulation by 4 Aspergillus carbonarius strains isolated from Spanish and Tunisian grapes were studied on grape-like medium. The limits of growth and OTA production have been identified. OTA production was significantly higher at 20 °C, followed by 25, 15, 30, 35 °C. No growth was observed at 7 °C and 42 °C after 10 d of incubation and, consequently, no OTA was detected at these temperature levels. In general, maximum mean OTA-producing capacity was found earlier with increasing incubation temperatures. However, at 35 °C, OTA was rarely detected although growth was maximum at this temperature. OTA accumulation was maximum after 10 d of incubation for all the temperatures except at 30 °C, were the maximum was detected at earlier incubation time (6 to 8 d) and then remained stable. Colony diameters and OTA accumulation along time were modeled by the Gompertz sigmoidal model and an estimation of maximum OTA accumulation rate and the delay until OTA production could be established. At high temperatures OTA accumulation starts earlier but increases slowly and becomes constant in a few days.     

Effect of pH on ochratoxin A production by Aspergillus niger aggregate species

The effect of pH (2-10) on growth and ochratoxin A (OTA) production by 12 Aspergillus niger aggregate strains was studied in two culture media: Czapek yeast autolysate agar (CYA) and yeast extract sucrose agar (YES), over 30 days. The strains were selected to include different sources, different reported abilities to produce OTA and different ITS-5.8S rDNA RFLP patterns. YES was a better culture medium than CYA for OTA production. In this medium, OTA was produced from pH 2 or 3 to 10 depending on the strain. The results show the ability of A. niger aggregate strains not only to grow, but also to produce OTA over a wide pH range. The results will lead to a better understanding of the role of A. niger aggregate strains in the OTA contamination of several food commodities.     

Contamination by moulds of grape berries in Slovakia

This paper describes the first map, albeit partial, of toxigenic fungi re-isolated from grape berries collected in three out of the six most important Slovakia winemaking areas in two different periods of the harvest year 2008. Low temperatures and high relative humidity during July 2008 favoured the development of grape fungal diseases that cause rots such as Plasmopara, Uncinula, Botrytis, Metasphaeria, Elsinoë, and Saccharomycetes. In the analysed samples, the following genera of toxigenic fungi were identified in the range of 1–4%: Aspergillus, Alternaria, Cladosporium, Epicoccum, Fusarium, Penicillium, Rhizopus, Ulocladium, and Trichoderma Trichothecium, while the genera Aspergillus, Alternaria, Fusarium, and Penicillium were in the range 11–29%. A. niger, A. carbonarius, some strains of A. carbonarius–with ‘crystals’ and strains of A. uvarum–uniseriate were identified; these species are considered ochratoxigenic (able to produce variable amounts of toxins). In addition, a non-ochratoxigenic strain of A. ibericus and a Fusarium strain able to biosynthesize small amount of fumonisins, beauvericin, and enniatins were identified. P. expansum, able to produce citrinin, represents 29.7%, of the Penicillium genus together with P. verrucosum, P. glabrum, P. citrinum, and P. crustosum. An analysis for the identification and quantification of the main toxins: ochratoxin A, fumonisins, beauvericin, enniatins, and fusaproliferin was performed on grape samples; it was consistent with the results of the mycological analysis. Toxigenic fungi should be checked throughout the years and their occurrence compared with all environmental factors to avoid health risks.     

黑曲霉产赭曲霉毒素A合成培养基的设计及产毒优化

针对目前没有适合黑曲霉产赭曲霉毒素A（OTA）全合成培养基的现状,进行黑曲霉产OTA合成培养基的设计,并优化高产OTA培养条件,以期为黑曲霉产OTA代谢和产毒机制的研究提供基础。通过碳源、氮源的筛选确定黑曲霉产OTA合成培养基的成分,利用高效液相色谱-荧光检测法检测此培养基在不同时间、pH、温度条件下OTA的产量,确定产OTA最佳培养条件。结果表明,培养基成分为FeSO4·7H2O 0.01 g/L,MgSO4·7H2O 0.5 g/L,Na2HPO4·2H2O 0.5 g/L,KCl 1.0 g/L,葡萄糖100 g/L,苯丙氨酸0.3 g/L。培养条件为初始pH 值为7,培养温度25 ℃,培养时间8 d。在此条件下黑曲霉产OTA产量最高,为4.19 μg/g,菌体生物量为3.4 g。     

葡萄中产赭曲霉毒素A炭黑曲霉的控制方法

炭黑曲霉(Aspergillus carbonarius)属曲霉属黑色曲霉菌,是葡萄中赭曲霉毒素A(ochratoxin A,OTA)主要产生菌,广泛存在于许多国家和地区的葡萄及其制品中,是造成葡萄酒中OTA污染的主要来源。控制这些霉菌的生长与产毒是从源头上减少葡萄原料及其制品OTA污染的关键环节,有关炭黑曲霉的污染规律及其控制方法方面的研究成为近年来的国际研究热点。根据已有研究,紫外线照射、杀菌剂、二氧化硫、纳他霉素、多酚、香精油、天然提取物和挥发性化合物等都能够在一定条件下对炭黑曲霉的生长与产生OTA的能力产生一定的抑制作用。最新研究发现,酵母、细菌和一些非产毒真菌及其代谢产物也对炭黑曲霉的生长和OTA产生具有良好抑制作用,而且具有无毒无害,安全性好等优点,在实际应用中展示出很好的应用潜力,越来越受到相关科研工作者和应用者的重视。本文主要围绕上述研究内容,对国内外近年来的相关研究进展进行综述。     

Degradation of Ochratoxin A by Proteases and by a Crude Enzyme of Aspergillus niger

Ochratoxin A is a mycotoxin present in food commodities as cereals, wine, coffee, figs, dried vine fruits or beer and in feeds for animals. The enhancement of its conversion into ochratoxin α is considered to be a way to reduce its presence in the body and, therefore, its toxicity. In this paper we report the ability of several commercial proteases to hydrolyze ochratoxin A into ochratoxin α in different amounts. After an incubation period of 25 h., a significant hydrolytic activity at pH 7.5 for Protease A (87.3%), and for Pancreatin (43.4%) was detected. At pH 3.0, a weak hydrolytic activity was detected for Prolyve PAC (3%). None of the other commercial enzymes tested were able to hydrolyze ochratoxin A in the tested conditions. Also, the isolation of an enzyme extract from an Aspergillus niger strain with very strong ochratoxin A hydrolytic activity at pH 7.5 (99.8%) is reported. This activity is similar to the activity detected in Protease A. Data about the inhibition effect of ethylenediaminetetraacetic acid and phenylmethanesulfonyl fluoride on the involved hydrolytic enzymes showed that enzymes involved in ochratoxin A hydrolysis are metalloproteins.     

春草莓表面霉菌鉴定及其致腐能力研究

目的调查春草莓表面霉菌的总体状况,对主要致腐真菌进行致腐能力和产毒素研究,为草莓的保鲜及食用安全评价提供理论依据。方法对60份春草莓样品进行霉菌检测,分离霉菌采用ITS序列测序方法进行鉴定;利用高效液相色谱法及酶联免疫法对腐烂草莓及真菌发酵液中赭曲霉毒素A(ochratoxin A,OTA)和展青霉素(patulin,PAT)进行检测,对出现率较高的霉菌进行草莓复接试验,判断其致腐能力。结果健康草莓表面的霉菌计数对数值3.5~5.5的占样品总数的83%;毛霉菌属、枝孢菌属和青霉菌属在草莓中出现率较高;由这3类霉菌致草莓腐烂样品和其发酵液均检测不出真菌毒素OTA和PAT,复接试验中毛霉属真菌在36 h在草莓表面形成明显菌斑,72 h可形成明显菌斑而更大面积软腐。结论草莓表面存在大量且多种霉菌,霉菌侵染草莓是导致其腐烂主要诱因,毛霉属霉菌占主要生态位但青霉菌属、枝孢菌属霉菌更易导致其软腐,草莓表面霉菌不会产生真菌毒素而对草莓进行二次污染。     

Identification of Aspergillus tubingensis strains responsible for OTA contamination in grapes and wine based on the acyl transferase domain of a polyketide synthase gene

Restriction digestion analysis of the acyl transferase (AT) domain sequences of a polyketide synthase (PKS) gene was tested as a rapid method to identify isolates of Aspergillus tubingensis from grapes. Restriction endonuclease digestion of PKS products using the endonucleases Bcc I, Hae III, Hpa II, Mbo I and Taq I distinguished five types of restriction fragment length polymorphism (RFLP). Ochratoxigenic isolates were only identified within RFLP-types I and III. The RFLP assay is proposed as a rapid and easy method to identify A. tubingensis isolates from grapes. Amino acid sequences of AT domains from representative A. tubingensis isolates of the RFLP types obtained were aligned and analysed using phylogenetic methods. A comparison was also made with reference strains of Aspergillus section Nigri . Most of the A. tubingensis strains clustered into two distinct groups Gr1 and Gr2 with the exception of two isolates that remained unclustered. These results support the intraspeficific variability within A. tubingensis species reported using other techniques.     

Effects of selected natural preservatives on the mycelial growth and ochratoxin A production of the food-related moulds Aspergillus westerdijkiae and Penicillium verrucosum

ABSTRACT

The present study examines the influence of the natural preservatives carvacrol, eugenol, trans-cinnamaldehyde and the essential oil (EO) Origanum vulgare on ochratoxin A (OTA) production and the mycelial growth of two food-related moulds, Penicillium verrucosum and Aspergillus westerdijkiae, by broth macro-dilution assay for 21 days. With the addition of ½ minimum inhibitory concentration (MIC) carvacrol, eugenol and O. vulgare EO, the mycelial dry weight of both moulds decreased significantly over the whole incubation period of 7, 14 and 21 days. Trans-cinnamaldehyde slightly stimulated the growth of A. westerdijkiae and P. verrucosum at 14 and 21 days of incubation. Growth inhibition did not accompany inhibition of OTA production. Although the growth of both moulds was inhibited after the addition of ½ MIC carvacrol, eugenol and O. vulgare EO, the OTA production of the strong mycotoxin producer A. westerdijkiae was stimulated. Only trans-cinnamaldehyde inhibited the production of OTA with the addition of ½ MIC. P. verrucosum produced significantly less OTA than A. westerdijkiae, and its mycotoxin production was almost completely inhibited by the addition of ½ MIC of the natural preservatives.     

新疆葡萄酒中赭曲霉毒素A含量分析

赭曲霉毒素A（OTA）是葡萄及其深加工产品中主要的真菌毒素,同时也被国际癌症研究机构（IARC）定为2B类致癌物。 采用 酶联免疫法（ELISA）商业试剂盒对新疆四大产区葡萄酒中OTA含量进行调研分析,研究不同产区葡萄酒中OTA含量的差异;同时, 对不同葡萄品种酿造葡萄酒过程中发酵葡萄汁和葡萄酒的OTA含量进行测定,分析酿造过程中OTA的变化规律。 结果显示,34份葡 萄酒样品中OTA含量均未超过2 μg/L;其中,焉耆盆地和吐哈盆地葡萄酒中OTA含量较低,平均含量分别为0.19 μg/L、0.20 μg/L;在 白葡萄酒酿造过程中OTA含量呈显著的下降趋势,而红葡萄酒酿造中OTA含量呈先升高后降低的趋势。     

Simultaneous detection of the main black aspergilli responsible for ochratoxin A (OTA) contamination in grapes by multiplex real-time polymerase chain reaction

This paper reports a duplex real-time polymerase chain reaction (PCR) assay for the simultaneous detection of members of the Aspergillus niger aggregate and A. carbonarius, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the β-ketosynthase and the acyl transferase domains of the poliketide synthase of A. carbonarius and the A. niger aggregate, respectively. Melting curve analysis using a SYBR Green I real-time PCR approach showed characteristic T _m-values demonstrating the specific, efficient and balanced amplification of the two PCR fragments. Subsequently, a TaqMan real-time PCR approach was settled, using 6-carboxy-fluorescein group (FAM) and VIC®-labelled specific probes for automated detection. Results indicated no differences in sensitivity when using either the two sets of primers and probes in separate or in the same reaction. However, when both targets are in very different amounts, there is a preferential amplification of the target which is in more concentration. CT-values obtained in the presence of grape DNA were very similar to those observed when only fungal purified DNA was present, indicating that the grape DNA does not interfere in the real-time PCR reaction. This procedure provides a fast and accurate tool to monitor, in a single reaction, the presence of OTA-producing species in grapes which, to some extent, will facilitate OTA contamination surveys to guarantee food safety in the wine industry.     

Ochratoxin A and the occurrence of ochratoxin A‐producing black aspergilli in stored peanut seeds from Córdoba,Argentina

Since there is no available information about the natural occurrence of ochratoxin‐producing fungi and ochratoxin A (OTA) in peanut seeds in Argentina, the aims of this work were to isolate and identify Aspergillus section Nigri and to evaluate the natural occurrence of OTA in stored peanut seeds. Likewise, the capacity to produce OTA by Aspergillus section Nigri was studied. Forty‐seven samples of peanut seeds were obtained from a storage plant in the south of Córdoba Province, Argentina. Each sample of 100 seeds was surface‐disinfected and plated onto a dichloran 18% glycerol agar (DG18). OTA was detected by HPLC. The production of OTA in strains belonging to section Nigri was tested in YES medium (2% yeast extract, 15% sucrose). Aspergillus spp., the most frequent mould, occurred in 100% of samples, followed by Penicillium spp. (87%) and Eurotium spp. (6.4%). A. flavus was isolated in 100% of the samples, followed by A. niger var. niger, A. niger var. awamori and A. carbonarius at a lower frequency, 89%, 68% and 4%, respectively. OTA was found in 32% of the peanut seed samples, with mean levels ranging from 0.5 to 170 ng g^?1. The mean recovery of the method used was 85 ± 2%. Forty‐three isolates (27%) of Aspergillus section Nigri, were found to be OTA producing strains. The highest percentage of ochratoxigenic strains (57%) was found within the A. carbonarius ssp. These results indicate a human exposure to OTA in Argentina through the ingestion of peanut seeds and peanut products. Copyright © 2006 Society of Chemical Industry     

Influence of water activity and temperature on conidial germination and mycelial growth of ochratoxigenic isolates of Aspergillus ochraceus on grape juice synthetic medium. Predictive models

The first stages in the development of Aspergillus ochraceus, an ochratoxin A‐producing fungus that infects grapes and may grow on them, have been studied on a synthetic nutrient medium similar to grape in composition. Spore germination and mycelial growth have been tested over a water activity (a_w) and temperature range which could approximate to the real conditions of fungal development on grapes. Optimal germination and growth were observed at 30 °C for all three isolates tested. Maximal germination rates were detected at 0.96–0.99 a_w at 20 °C, while at 10 and 30 °C the germination rates were significantly higher at 0.99 a_w. Although this abiotic factor (a_w) had no significant influence on mycelial growth, growth rates obtained at 0.98 a_w were slight higher than those at other a_w levels. Predictive models for the lag phase before spore germination as a function of water activity and temperature have been obtained by polynomial multiple linear regression, and the resulting response surface models have been plotted. Copyright © 2005 Society of Chemical Industry     

不同条件对炭黑曲霉产赭曲霉毒素A能力的影响

为了解一株分离自新疆葡萄园的炭黑曲霉菌株在不同培养条件下产生赭曲霉毒素A(OTA)的能力,试验采用孟加拉红培养基,利用高效液相-荧光法对OTA进行定量检测,在单因素试验的基础上,采用响应面法优化产毒条件。结果表明,对该菌株产生OTA的条件影响从大到小依次为葡萄糖质量浓度、乙醇体积分数、SO2质量浓度。最优产毒条件:在温度28℃条件下,葡萄糖质量浓度92.26g/L、SO2质量浓度8.26mg/L、乙醇体积分数0.05%。在此条件下,OTA产量为7.427ng/mL。     

控制葡萄及其制品中赭曲霉毒素A的研究进展

赭曲霉毒素A(OTA)是一种有毒的真菌次级代谢产物,在葡萄及其制品中经常被检出。本文综述了控制葡萄及其制品中OTA的主要措施,包括抑制OTA产生菌的生长及其产毒和去除已产生的OTA两方面。葡萄中的致病菌主要是青霉和曲霉,而炭黑曲霉是其中最主要OTA产生菌,通过模拟葡萄生长及葡萄制品的生产过程,发现温度、光照、湿度等因素是影响OTA产生菌的生长及其产毒的重要因素。去除OTA的方法概括的分为物理方法、化学方法、生物方法,其中生物降解OTA是目前研究的热点。      

Occurrence of fruit rot fungi (Aspergillus section Nigri) on some drying varieties of irrigated grapes

The occurrence of Aspergillus section Nigri on certain varieties of grapes used for dried vine fruit (and especially on Sultana), was surveyed extensively during three harvest seasons (1998, 1999 and 2000). Members of Aspergillus section Nigri were enumerated and identified in a total of 806 samples comprising both fresh fruit as well as partially-dried and fully-dried grapes. Aspergillus aculeatus , A. carbonarius and A. niger were commonly isolated. Of those three species, A. niger showed the highest optimum temperature for growth, as well as highest thermal tolerance. That combination of properties probably accounts for the occurrence of A. niger in over 80% of fruit samples in all three seasons. A. carbonarius was also prevalent on fruit surveyed for fungal rots. Severity of infection with this group of fungi was highest in the seasons when grape berries were rain-damaged. Typically, the Aspergillus count increased during the initial stages of drying. Growth of these moulds while grapes are either still on the vine or during drying carries important implications for human consumption due to the potential for certain strains of these fungi to produce ochratoxin A. Fungal cultures on coconut cream agar plates were screened for production of ochratoxin A via fluorescence emission under UV light. Such fluorescence was observed in all isolates of A. carbonarius , but was not observed in any isolates of either A. aculeatus or A. niger .