Search

Dothideomycetesis the largest class of kingdom Fungi and comprises an incredible diversity of lifestyles, many of which have evolved multiple times. Plant pathogens represent a major ecological niche of the class Dothideomycetesand they are known to infect most major food crops and feedstocks for biomass and biofuel production. Studying the ecology and evolution of Dothideomyceteshas significant implications for our fundamental understanding of fungal evolution, their adaptation to stress and host specificity, and practical implications with regard to the effects of climate change and on the food, feed, and livestock elements of the agro-economy. In this study, we present the first large-scale, whole-genome comparison of 101 Dothideomycetesintroducing 55 newly sequenced species. The availability of whole-genome data produced a high-confidence phylogeny leading to reclassification of 25 organisms, provided a clearer picture of the relationships among the various families, and indicated that pathogenicity evolved multiple times within this class. We also identified gene family expansions and contractions across the Dothideomycetesphylogeny linked to ecological niches providing insights into genome evolution and adaptation across this group. Using machine-learning methods we classified fungi into lifestyle classes with >95 % accuracy and identified a small number of gene families that positively correlated with these distinctions. This can become a valuable tool for genome-based prediction of species lifestyle, especially for rarely seen and poorly studied species.

Some species of Fusariumcan produce mycotoxins during food processing procedures that facilitate fungal growth, such as the malting of barley. The objectives of this study were to develop a 5′ fluorogenic (Taqman) real-time PCR assay for group-specific detection of trichothecene- and fumonisin-producing Fusariumspp. and to identify Fusarium graminearumand Fusarium verticillioidesin field-collected barley and corn samples. Primers and probes were designed from genes involved in mycotoxin biosynthesis (TRI6and FUM1), and for a genus-specific internal positive control, primers and a probe were designed from FusariumrDNA sequences. Real-time PCR conditions were optimized for amplification of the three products in a single reaction format. The specificity of the assay was confirmed by testing 9 Fusariumspp. and 33 non- Fusariumfungal species. With serial dilutions of purified genomic DNA from F. verticillioides, F. graminearum, or both as the template, the detection limit of the assay was 5 pg of genomic DNA per reaction. The three products were detectable over four orders of magnitude of template concentration (5 pg to 5 ng of genomic DNA per reaction); at 50 ng template per reaction, only the TRI6 and FUM1 PCR products were detected. Barley and corn samples were evaluated for the presence of Fusariumspp. with traditional microbiological methods and with the real-time PCR assay. The 20 barley samples and 1 corn sample that contained F. graminearumby traditional methods of analysis tested positive for the TRI6 and internal transcribedspacer (ITS) PCR products. The five corn samples that tested positive for F. verticillioidesby traditional methods also were positive for the FUM1 and ITS PCR products. These results indicate that the described multiplex real-time PCR assay provides sensitive and accurate differential detection of fumonisin- and trichothecene-producing groups of Fusariumspp. in complex matrices.

The genus Fusariumcomprises a diverse group of fungi including several species that produce mycotoxins in food commodities. In this study, a multiplex polymerase chain reaction (PCR) assay was developed for the group-specific detection of fumonisin-producing and trichothecene-producing species of Fusarium. Primers for genus-level recognition of Fusariumspp. were designed from the internal transcribed spacer regions (ITS1 and ITS2) of rDNA. Primers for group-specific detection were designed from the TRI6gene involved in trichothecene biosynthesis and the FUM5gene involved in fumonisin biosynthesis. Primer specificity was determined by testing for cross-reactivity against purified genomic DNA from 43 fungal species representing 14 genera, including 9 Aspergillusspp., 9 Fusariumspp., and 10 Penicilliumspp. With purified genomic DNA as a template, genus-specific recognition was observed at 10 pg per reaction; group-specific recognition occurred at 100 pg of template per reaction for the trichothecene producer Fusarium graminearumand at 1 ng of template per reaction for the fumonisin producer Fusarium verticillioides. For the application of the PCR assay, a protocol was developed to isolate fungal DNA from cornmeal. The detection of F. graminearumand its differentiation from F. verticillioideswere accomplished prior to visible fungal growth at <105CFU/g of cornmeal. This level of detection is comparable to those of other methods such as enzyme-linked immunosorbent assay, and the assay described here can be used in the food industry's effort to monitor quality and safety.