Medina Angel, Mateo Rufino, López-Ocaña Laura, Valle-Algarra Francisco Manuel, Jiménez Misericordia
Departamento de Microbiología y Ecología, Facultad de Biología, Universidad de Valencia, Dr. Moliner 50, E-46100 Burjasot, Valencia, Spain.
Appl Environ Microbiol. 2005 Aug;71(8):4696-702. doi: 10.1128/AEM.71.8.4696-4702.2005.
The native mycobiota of five grape varieties grown in Spain has been studied. Four (Bobal, Tempranillo, Garnacha, and Monastrell) were red varieties and one (Moscatel) was white. The main fungal genera isolated were Alternaria, Cladosporium, and Aspergillus. The isolation frequency of Aspergillus spp. section Nigri in contaminated samples was 82%. Ochratoxin A (OTA) production was assessed using yeast extract-sucrose broth supplemented with 5% bee pollen. Cultures of 205 isolates from this section showed that 74.2% of Aspergillus carbonarius and 14.3% of Aspergillus tubingensis isolates produced OTA at levels ranging from 1.2 to 3,530 ng/ml and from 46.4 to 111.5 ng/ml, respectively. No Aspergillus niger isolate had the ability to produce this toxin under the conditions assayed. Identification of the A. niger aggregate isolates was based on PCR amplification of 5.8S rRNA genes and its two intergenic spacers, internal transcribed spacer 1 (ITS1) and ITS2, followed by digestion with restriction endonuclease RsaI of the PCR products. The restriction patterns were compared with those from strains of A. niger CECT 2807 and A. tubingensis CECT 20393, held at the Spanish Collection of Type Cultures. DNA sequencing of the ITS1-5.8S rRNA gene-ITS2 region of the OTA-producing isolates of A. tubingensis matched 99 to 100% with the nucleotide sequence of strain A. tubingensis CBS 643.92. OTA determination was accomplished by liquid chromatography with fluorescence detection. OTA confirmation was carried out by liquid chromatography coupled to ion trap mass spectrometry. The results showed that there are significant differences with regard to the isolation frequency of ochratoxinogenic fungi in the different grape varieties. These differences were uncorrelated to berry color. The ability of A. tubingensis to produce OTA and the influence of grape variety on the occurrence of OTA-producing fungi in grapes are described in this report for the first time.
对西班牙种植的五个葡萄品种的本地真菌群落进行了研究。其中四个（博巴尔、丹魄、歌海娜和莫纳斯特雷尔）是红葡萄品种，一个（麝香葡萄）是白葡萄品种。分离出的主要真菌属为链格孢属、枝孢属和曲霉属。曲霉属黑曲霉组在受污染样品中的分离频率为82%。使用添加5%蜂花粉的酵母提取物 - 蔗糖肉汤评估赭曲霉毒素A（OTA）的产生。来自该组的205个分离株的培养表明，74.2%的黑曲霉和14.3%的管曲霉分离株产生OTA，水平分别为1.2至3530 ng/ml和46.4至111.5 ng/ml。在测定条件下，没有黑曲霉分离株有产生这种毒素的能力。黑曲霉聚集体分离株的鉴定基于5.8S rRNA基因及其两个基因间隔区（内部转录间隔区1（ITS1）和ITS2）的PCR扩增，随后用限制性内切酶RsaI消化PCR产物。将限制性图谱与保存在西班牙模式培养物保藏中心的黑曲霉CECT 2807菌株和管曲霉CECT 20393菌株的图谱进行比较。管曲霉OTA产生分离株的ITS1 - 5.8S rRNA基因 - ITS2区域的DNA测序与管曲霉CBS 643.92菌株的核苷酸序列匹配度为99%至100%。OTA的测定通过液相色谱 - 荧光检测完成。OTA的确证通过液相色谱 - 离子阱质谱联用进行。结果表明，不同葡萄品种中产赭曲霉毒素真菌的分离频率存在显著差异。这些差异与浆果颜色无关。本报告首次描述了管曲霉产生OTA的能力以及葡萄品种对葡萄中OTA产生真菌发生的影响。
