High Fumonisin Content in Maize: Search for Source of Infection and Biological Function
by Raana Dastjerdi
Date of Examination:2014-05-08
Date of issue:2014-05-22
Advisor:Prof. Dr. Petr Karlovsky
Referee:Prof. Dr. Petr Karlovsky
Referee:Dr. Horst-Henning Steinmann
Referee:Prof. Dr. Andreas von Tiedemann
Persistent Address:
http://dx.doi.org/10.53846/goediss-4519
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Abstract
English
Fusarium is a large and complex genus, comprises important pathogens most of which are able to produce mycotoxins. Maize is a host of several Fusarium species that cause ear rot, kernel rot, stalk rot and also seedling blight. Fusarium contamination of kernels and maize products had always been a serious concern. Owing to the positive correlation between fungal biomass and mycotoxin content, quantification of fungal DNA in plant materials would be considered as an initial fast and cost-effective mean to evaluate the risk of grain contamination. Several quantification methods are well-known of which real-time PCR (qPCR) has been used as an effective tool for species-specific quantification of fungal biomass in plant tissues. The method mostly relies on standard thermocyclers (96-well or 384-well blocks) with a separate run for each template and usually set in total reaction volume of 15 to 25 µl. In this investigation, we developed a multi-species qPCR assay for simultaneous quantification of genomic DNA of the nine Fusarium species with 384-well microplates in a total volume of 4 µl. The sensitivity of the method ranged from 0.05 - 1.52 pg DNA per reaction, and the repeatability ranged from 0.81% to 1.71% RSD. Developed low volume qPCR assay was successfully employed for the analysis of weed plants to the infection of nine Fusarium spp. The main objectives were determination the role of weeds in the survival of maize fusarium pathogens and assessment of their ability for producing of the main mycotoxins. The Real-time PCR detected eight Fusarium species in 201 weed samples representing 36 weed species, collected from maize fields. The highest frequency was observed for F. equiseti (49%) and F. avenaceum (34.7%). Similar diversity of Fusarium spp. was observed in both conventional and organic farming systems. Isolation of Fusarium strains from 12 common weed species were carried out, the strains were identified based on the morphological characters and then identification was confirmed by using the translation elongation factor 1-alpha (TEF-1α) gene sequence. The recovery rate was high for F. equiseti (32.7%) and F. avenaceum (21%). None of the field samples as well as weed plants tested in inoculation studies show obvious symptoms of Fusarium infection. Re-isolation of the strains from artificially inoculated plants confirmed the endophytic infection of weeds by Fusarium spp. The present study reports five new alternative hosts for Fusarium species in maize fields. High incidence rate of beauvericin and enniatins contamination was obtained in weed samples while trichothecenes, fumonisins and zearalenone were not detected in any of the weeds studied. Although toxicity of fumonisins to plants and field animals has been clearly demonstrated, the function of this toxin, however, in virulence of F. verticillioides toward maize plants is still unknown. In present study, virulence of two non-fumonisins producing strains (fum 1-3 and fum 1-4) and their progenitors (FUM 1-1) was assessed on different plants including maize, sorghum, rice and beetroot seedlings grown under greenhouse conditions. The quantity of fungal biomass in plant tissues was considered as an indicator of fungal aggressiveness and it was measured by the developed low volume qPCR protocol. There was no significant (P = 0.05) differences between each wild type and the relevant mutant for colonization of plant tissues. In inoculated maize, rice and beetroot seedlings, systemic fungal infection was observed from roots to the aboveground parts; but rate of systemic transmission was low in sorghum plants. Although our results are not enough to make a final conclusion of fumonisin function in virulence of F. verticillioides on rice and sorghum seedlings, however, the results show diseases incidence was independent of fumonisins production in maize seedlings. In our system, therefore, fumonisins had no role as a pathogenicity factor.
Keywords: Fusarium spp.,Weed plants, Fumonisin, Biological function