Studies On Multiplex PCR Detection Method And Toxicity Of Fumonisin-producing Strains In Corn And Asparagus

Fumonisins are a group of mycotoxins mainly produced by Fusahum vertici-llioides (formerly F. moniliforme) and Fusarium proliferatum. Fumonsins have been demonstrated to cause several fatal diseases in animals, such as leukoencephalomala-cia in horses, pulmonary oedema and hydrothorax in swine and hepatic cancer in rats. In humans, fumonisins are associated with oesophageal cancer and birth neural tube defects. F. verticillioides and F. proliferatum are found worldwide and have been reported in maize, asparagus, and many other agriculture products.We have developed an economical, rapid, simple, and precise multiplex PCR method for identifying fumonsin-producing fungal strains in corn and asparagus. We have also developed a method for direct quantification of B-series fumonsins (FBs) in corn products. This method is based on high-performance liquid chromatography coupled with an evaporative laser scattering detector (HPLC- ELSD), which allows direct detecting the non-UV-absorbing fumonisins without any prior derivatization of the samples. The method ensures accurate quantification of FBs with a limit of 6 ng/μl.Using the methods, we have studied the occurrence of fumonsin-producing strains in maize grain samples from household and supermarket of limited areas in China and in edible asparagus from Zhejiang Province. Furthermore, the ability of the isolated srains to produce FBs on sterile maize grain has been determined. The results of the studies are as follows:Several pairs of primers that are specific for fumonsin-producing strains were designed. These primers, including fuml-2L/fuml-2R, fum8-3N/fum-8-4E, fum17-P-F2/fuml7-P-R2, rp32/rp33, and rp679/ rp680 (Proctor et al., 2004), were based on the polykefide synthase (PKS) gene (FUMI), serine-palmitoyltransferase gene (FUM8), and longevity assurance factor (also known as ceramide synthase) gene (FUMI7) that are involved in fumonisin biosynthesis. In addition, Fusarium genus-specific primers, ItsR/ItsF (Bluhm et al., 2002), were also prepared. With both fumonisin-specific primers and genus-specific primers, Multiplex PCR were carried out to detect fumonsin-producing strains and Fusarium verticllioides in the food products collected from various areas in China. As control experiments, the wild-type fumonisin-producing strain A0149 (FGSC NO.7600) of F. verticillioides, the PKS-deleted non-fumonisin-producing mutant, and Penicillium citrinum were included in the multiplex PCR. The detection limits of the multiplex PCR detecting fumonisin-producing Fusarium verticillioides with primers fuml-2R, fum8-3N/fum-8-4E, fum17-P-F2/fum17-P-R2 and ItsR/ItsF and the multiplex PCR detecting fumonisin-producing strains with primers rp32/rp33, rp679/rp680 and ItsR/ItsF were 1 ng and 100 pg template DNA per PCR assay respectively.Among 58 corn samples, 21 samples contained fungal strains (total 25 isolates); from 16 edible asparagus samples, 16 isolates were identified. The 41 isolates were analyzed with the multiplex PCR, and the results showed that 25 were fumonisin-producing Fusarium strains (15 from corn and 10 from asparagus). The positive strains were subjected to further morphological identification, as well as molecular identification by analyzing the sequence of PCR amplified translation elongation factor-1α(TEF). The results showed that all the isolates from corn are F. verticillioides and all the isolates from asparagus were F proliferatum.The ability of the positive isolates identified by PCR to produce fumonisins was examined by HPLC-ELSD. All 25 isolates produced FB₁ (125-40321μg/g) on autoclaved maize kernels. The 10 asparagus isolates were all high producer (≥500μg/g) except one isolate with a modest yield (50μg/g-500μg/g). The ability to produce FBs differed among different isolates of the same species. For the F. verticillioides isolates, the yield of FB₁ varied from 1331 to 40321μg/g, while for F. proliferatum isolates the yield of FB₁ from 125 to 19251μg/g. Most isolates also produced FB₂ and FB₄, while some also produced FB₃. In all isolates, the yield of FB₁ was always the highest among all fumonisins.