Studies On Fumonisins Contamination Of Corn And Asparagus Products And The Resistance Of Arabidopsis To FB₁

Fumonisins (FB) are a group of mycotoxins produced by Fusarium spp.includingFusarium verticillioides and Fusarium proliferatum.Due to the structural similaritywith sphingolipids,fumonisins can cause diseases in animals and plants.Fumonsinshave been demonstrated to cause several fatal diseases in animals,such asleukoencephalomalacia in horses,pulmonary oedema and hepatic cancer in rats.Inhumans,fumonisins are associated with oesophageal cancer and birth neural tubedefects.Fumonisin contamination occurs ubiquitously-it was found in manyagricultural products,such as corn,corn products and asparagus spears,which isassociated with food safety,livestock loss and human health risks.Fumonisin B1 (FB₁)induces programmed cell death (PCD) and hypersensitive reaction (HR) in plant,providing a good system to study innate immune response,chemical stress andsphinglipids function in Arabidopsis thaliana (Arabidopsis).In present study,aneffective high-performance liquid chromatography coupled with an evaporative laserscattering detector (HPLC-ELSD) method was developed for detection of fumonisinsin food products.The method was used for the analysis of fumonisins contamination incorn products from different areas including a high-risk area for esophageal cancer.The fumonisins level was also assessed by LC-MS detection in asparagus spears fromZhejiang Province.Using PCR and fumonisin-producing culture analysis,the factorscausing fumonisin production in corn and asparagus spears were also discussed.Inaddition,two Arabidopsis FB₁-resistant mutants were isolated,and the role ofglucosinolates in FB₁-induced PCD was studied in Arabidopsis.Following are themain results.A new HPLC-ELSD method was developped for detection and quantification offumonisins in foods without any prior derivatization of the samples,it is simple andfast comparing to the conventional OPA derivatization method.We analyzed thefumonisins in corn-based food samples from central markets in eastern China byHPLC-ELSD method.The results showed that FB₁ was the main contaminant in thesamples.The overall level of fumonisin contamination was relatively low,with a range of 0.25 tol.80μg/g (mean 0.74μg/g) in 66.7% (16 of 24) of corn samples fromMiddle-eastern Area,0.21 to 0.29μg/g (mean 0.24μg/g) in 28.6% (6 of 21) of cornsamples from Northeastern Area,and 0.30 to 3.13μg/g (mean 0.47μg/g) in 30.0% (6of 20) of corn samples from Southeastern Area.The level of mycotoxin fumonisins in corn-based food and feed collected fromLinxian County,a high-risk area for esophageal cancer in China,was analyzed byHPLC-ELSD.A total of 104 corn kernel samples was obtained from local household,granary,wholesale market (central market),and retail markets (store and supermarket).Fumonisin B1 was detected in the samples from household,granary,central market,and store,with a positive rate of 61.5%,50%,33.3%,and 17%,respectively.Nofumonisin was detected in samples from supermarket.The highest FB₁ levels (0.30 to3.20μg/g;mean,1.42μg/g) were found in samples from granary,followed byhousehold (0.25 to 1.80μg/g;mean,0.73μg/g),central market (0.25 to 1.10μg/g;mean,0.51μg/g),and store (0.22 to 0.34μg/g;mean,0.28μg/g).Among the 80 cornkernel samples collected from local household,18 of 24 (75.0%) moldy samplescontained high levels of FB₁ (0.28 to 3.30μg/g;mean,1.58μg/g),and 20 of 56(35.7%) apparently healthy samples contained low levels of FB₁ (0.21 to 0.82μg/g;mean,0.46μg/g).As central market plays an important role in trade of corn-basedfood and feed in China,a total of 115 corn-based food and feed samples were collectedfrom local central market.The highest FB1 levels (0.30 to 3.13μg/g;mean,1.50μg/g)were found in feed,followed by unprocessed food (0.31 to 0.63μg/g;mean,0.47μg/g)and processed food (0.21 to 0.28μg/g;mean,0.25μg/g).The positive incidence of FB₁in feed,unprocessed,and processed food were 53.6%,33.3% and 17.9%,respectively.In conclusion,the results show that corn-based food and feed from Linxian Countycontained low level of FB₁ (＜2μg/g) in general,but efforts should be made to controlthe fumonisin contamination in corn kernels stored in granary and household.In the survey to elucidate the relationship between fumonisin-producing fungi andfumonisin contamination in corn,morphology characterization and PCR detectionwere conducted to identifying Fusaiusm spp.Isolates,whose capacity of producingfumonisins were then analyzed by inoculating cracked maize kernels (CMK) medium. Generally,the contamination rate of fungi,Fusarium spp.and fumonisin-producingfungi is 42.18%,35.94% and 29.69%,respectively,and the proportion of Fusariumand fumonisin-producing fungi in all contamination isolates is 74.19% (23/31) and61.29% (19/31),respectively.Our results showed that three Fusarium speciesincluding F.versillioides,F.proliferatum and F.subglutinans were demonstrated tocontain FUM1 and FUM8 genes and also produce fumonisins in CMK medium.Fumonisins production tests indicated that the ability of fumonisin production variedsignificantly among different species or different strains within the same Fusariumspecie.The fumonisin level of fumonisin-producing isolates was from 186μg/g to16784μg/g (average,4637μg/g);FB₁ is the primary component and up to 4041μg/g inaverage.In addition,the occurrence of fumonisin-producing isolates was differentamong corn samples from the three different areas,the most serious fungi andfumonisin-prouducer contamination was found in samples from eastern area.Ourresearch also suggested that fumonisin contamination was detected even in clean andhealthy edible corns,the colonization of fumonisin-producer likely occurred inpre-harvest,but fumonisin-production did not occure due to the unsuitable conditionsduring post-harvest.When the environment changed,these high-fumonisin-producingstrains might produce fumonisin.toxins in infected corn samples.The results obtained from HPLC-ELSD and LC-MS analyses showed that theasparagus samples from different areas of Zhejiang Province did not contain adetectable level of fumonisins.All asparagus samples collected for this study wereapparently clean and healthy.However,approximately 70% of the samples werecontaminated with Fusarium,including F.proliferatum,F.oxysporum,F.acuminatumand F.equesti.Among the fungal isolates,F.proliferatum accounted for over 50%.ThePCR results showed that fungal isolates contained the FUM genes,and also producedfumonisins in cultures,ranging from 28-4204μg/g culture media.Although multipleFusarium species were identified,our results showed that F.proliferatum was the onlyfumonisin-producing contaminant in the asparagus samples,although other Fusariumspecies might produce other different class of mycotoxins.Despite the fact that theasparagus from Zhejiang Province do not have apparent fumonisin contamination,there is a potential risk for human consumption of the asparagus spears due to the high frequency of natural occurrence of Fusarium spp,which could produce toxicmycotoxins,such as fumonisins produced by F.proliferatum.Among 15000 ethyl methanesulfonic acid-mutagenized Arabidopsis Col-0 seedssubjected to selection on 1μM FB₁,two FB₁-resistant (for) mutantsfbr41 and for42,which exhibited the most penetrant phenotypes,were identified and characterized infurther detail.In addition to resistance to FB₁,the mutants also showed rich root hair,hard leaf and less reactive oxygen species (ROS) production when grew on the FB₁medium and the tolerance of both mutants are much stronger than those of jar1-1,ein3-1,nprl mutants and transgenic NahG plants.Moreover,they display altered plantarchitecture in the absence of FB₁,such as dwarf,enhanced leaf margin serration anddeveloped lateral roots.A chi-square goodness-of-fit test confirmed that the fbr41phenotype of the F2 progeny segregated at the expected 3:1 (sensitive:resistant) ratiofor a single recessive mutation.The glucosinolates content of leaves in the Arabidopsis wild-type Col-0 wereanalyzed by HPLC after leaf infiltration with 10μM FB₁.The results showed that thecontent of short-chain aliphatic glucosinolates decreased,while the content oflong-chain aliphatic glucosinolates increased.Similarly,the content of indol-3-ylmethylglucosinolate (IM) decreased,while the content of 4-methoxyindol-3-ylmethylglucosinolate (4IM) and 1-methoxyindol-3-ylmethylglucosinolate (1IM)increased significantly.The observations that 4IM and IM were also induced in bothglucosinolate mutants gcc8 and gsml-3,and gsml-3 displayed a better resistance toFB₁ than Col-0,indicated that 4IM and 1IM instead of long-chain aliphaticglucosinolates might be involved in the resistance of Arabidopsis to FB₁.Geneexpression analysis suggested that no significant difference was observed between FB₁treatment and control in expression of NIT2 and PAD3,which were involved in IAAand camalexin biosynthesis,respectively.On the contrary,CYP83B1 was greatlyup-regulated by FB₁ treatment.The content of 4IM and 1IM is higher in both fbrmutants than that of wild-type Col-0.These results indicated that the biosynthesis of4IM and IM might be related to the resistance to FB₁ in Arabidopsis.The mechanisminvolved remains to be further elucidated.