Department of Studies in Biotechnology, University of Mysore, Mysore, 570006, Karnataka, India.
J Sci Food Agric. 2014 Apr;94(6):1132-9. doi: 10.1002/jsfa.6380. Epub 2013 Oct 4.
Fusarium spp. are not only pathogenic to plants but are also known as toxin producers that negatively affect animal and human health. The identification of Fusarium spp. remains one of the most critical issues in fungal taxonomy. In this study, different strains of Fusarium spp. were isolated from sorghum seed samples and identified at the molecular level by tef-1α gene amplification. A multiplex polymerase chain reaction (mPCR) assay was developed to differentiate toxigenic and non-toxigenic Fusarium spp. by designing a primer for the Fum21 gene along with the Fum1 and Fum8 genes. A competitive direct enzyme-linked immunosorbent assay (CD-ELISA) was employed to assess the fumonisin-producing ability of Fusarium spp. Phylogenetic analyses were performed using partial sequences of tef-1α and inter-simple sequence repeat (ISSR) markers of different Fusarium spp.
All 27 isolates of Fusarium spp. were positive for the tef-1α gene and revealed the presence of F. verticillioides, F. thapsina and F. cf. incarnatum-equiseti complex. The standardized mPCR assay distinguished toxigenic and non-toxigenic F. verticillioides. Further, mPCR fumonisin-positive F. verticillioides isolates were also positive by CD-ELISA. The tef-1α gene sequence was found to be useful in revealing intraspecific polymorphism to some extent. ISSR markers revealed a high level of polymorphism among different isolates of Fusarium spp., and the dendrogram of ISSR analyses grouped the 27 isolates into two major clusters.
The present method provided rapid and reliable detection of fumonisin-producing Fusarium spp. The mPCR assay could be an alternative strategy to current conventional mycotoxin analytical techniques and a reliable tool for high-throughput monitoring of major mycotoxin-producing fungi during the processing steps of food and feed commodities.
镰刀菌不仅对植物具有致病性，而且还被认为是产生毒素的病原体，这些毒素会对动物和人类健康造成负面影响。镰刀菌的鉴定仍然是真菌分类学中最关键的问题之一。在本研究中，从高粱种子样本中分离出不同的镰刀菌菌株，并通过 tef-1α 基因扩增在分子水平上进行鉴定。设计了针对 Fum21 基因的引物，同时设计了针对 Fum1 和 Fum8 基因的引物，开发了多重聚合酶链反应（mPCR）检测方法，以区分产毒和非产毒镰刀菌。采用竞争直接酶联免疫吸附测定（CD-ELISA）评估不同镰刀菌的产伏马菌素能力。使用 tef-1α 的部分序列和不同镰刀菌的简单重复间序列（ISSR）标记进行系统发育分析。
27 株镰刀菌均扩增出 tef-1α 基因，且存在 F. verticillioides、F. thapsina 和 F. cf. incarnatum-equiseti 复合体。标准化 mPCR 检测方法可区分产毒和非产毒 F. verticillioides。此外，mPCR 伏马菌素阳性的 F. verticillioides 分离株也通过 CD-ELISA 检测呈阳性。tef-1α 基因序列在一定程度上有助于揭示种内多态性。ISSR 标记显示不同镰刀菌分离株之间存在高度多态性，ISSR 分析的聚类树状图将 27 个分离株分为两个主要聚类。
本方法可快速可靠地检测产伏马菌素的镰刀菌。mPCR 检测方法可能是当前常规真菌毒素分析技术的替代策略，也是在食品和饲料商品加工过程中对主要产毒真菌进行高通量监测的可靠工具。
