Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS668. Population shifts and mating-type heterokaryosis in Aspergillus flavus. Rodrigo A. Olarte 1 , Bruce W. Horn 2 , Carolyn J. Worthington 1 , Rakhi Singh 1 ,Ignazio Carbone 1 . 1) Department of Plant Pathology, North Carolina State University, Raleigh, NC; 2) National Peanut Research Laboratory, AgriculturalResearch Service, U.S. Department of Agriculture, Dawson, GA.Aspergillus flavus is a heterothallic fungal pathogen of many economically important crops worldwide. We sampled A. flavus strains from a cornfield inRocky Mount, North Carolina, USA. Plots were inoculated at tasselling with either A. flavus AF36 or NRRL 21882 (=Afla-Guard) nonaflatoxigenic biocontrolstrains, both of which are mating type MAT1-2. Subsequently, aflatoxigenic strain NRRL 3357 (MAT1-1) was applied to all plots, including control plots notinoculated with biocontrol strains. Sclerotia were harvested from infected corn ears and ninety single-ascospore isolates were obtained from ascocarpsoriginating from plots treated with AF36 and NRRL 21882. In addition, eighty A. flavus isolates were collected from soil one month after planting (beforebiocontrol application) and one year after biocontrol application, for a grand-total of 250 isolates. PCR amplification revealed grouping of isolates intothree distinct mating-type classes: MAT1-1, MAT1-2 and MAT1-1/MAT1-2. An overwhelming majority (54%) of isolates sampled prior to biocontroltreatments were heterokaryotic for mating type (MAT1-1/MAT1-2), but was shifted to only 9% of isolates from soil after biocontrol treatments; 39% ofisolates obtained from ascospores were heterokaryotic, with the remaining comprising either MAT1-1 or MAT1-2. Multilocus genotyping indicated thatascospores might have originated from Afla-Guard as a putative parent; there was no evidence of AF36 or NRRL 3357 in ascospores or in pre- or posttreatmentsoil samples, which may explain the genetic structure of the indigenous population. The vertical transmission of MAT1-1/MAT1-2 to progenyascospore isolates suggests that heterokaryosis can be maintained in subsequent generations. Furthermore, matings were performed to determinefunctionality of these MAT1-1/MAT1-2 strains and all isolates tested were strictly functional as MAT1-2. Further characterization of heterokaryons andtheir frequency in A. flavus populations may be important in understanding the adaptation of these fungi to changing environmental conditions and couldlead to better and more effective biocontrol strategies specific to a geographic region. Understanding population structure is the key to unlocking thesecrets of a successful biocontrol strain.669. Structural variation of trichothecene mycotoxins has resulted from multiple evolutionary processes in the fungal order Hypocreales. R. H. Proctor 1 ,A. M. Stanley 1 , M. G. Malmierca 2 , N. J. Alexander 1 , S. Gutiérrez 2 , S.P. McCormick 1 . 1) Bacterial Foodborne Pathogens and Mycology, USDA ARS NCAUR,Peoria, IL; 2) Universitary School of Agricultural Engineers, University of León, Ponferrada, Spain.Trichothecenes are secondary metabolites produced by fungi in at least six genera of the order Hypocreales. These metabolites are of concern becausethey are toxic to humans and other animals and can accumulate in grain used for food and feed. They also contribute to plant pathogenesis of Fusariumand to biological control activity of Trichoderma. Although all trichothecenes share the same molecular skeleton, a tricyclic structure with a12,13-epoxide,different genera produce trichothecenes that differ in patterns of oxygenation and acylation. To investigate how such structural variation has evolved, weexamined 1) variation in gene function and content in homologs of the trichothecene biosynthetic gene (TRI) cluster and 2) phylogenetic relationships ofTRI genes among trichothecene-producing genera. The results suggest that the ancestral hypocrealean TRI cluster consisted of at least seven genes,including the enzyme-encoding genes TRI4 and TRI5 responsible for synthesis of the trichothecene skeleton, the regulatory genes TRI6 and TRI10, and thetransporter gene TRI12. Phylogenetic analyses indicate that oxygenation of carbon atom 4 (C-4), which occurs in all trichothecene-producing genera, likelyarose when different genera acquired different C-4 hydroxylase genes: e.g. TRI11b in Trichoderma and Myrothecium and TRI13 in Fusarium. In contrast, C-3 oxygenation, which occurs in only one genus, likely arose by a change in function of TRI4, a gene that exists in all genera. These results, and those fromstudies of Fusarium and Trichoderma, indicate that structural variation of trichothecenes has arisen by recruitment, changes in function, and deletion ofTRI genes during evolution of the Hypocreales.670. Evidence for birth-and-death evolution and horizontal transfer of the fumonisin mycotoxin biosynthetic gene cluster in Fusarium. R.H Proctor 1 , F.Van Hove 2 , A. Susca 3 , G. Stea 3 , M. Busman 1 , T. van der Lee 4 , C. Waalwijk 4 , A. Moretti 3 , T.J.. Ward 1 . 1) Bacterial Foodborne Pathogens and Mycology, USDAARS NCAUR, Peoria, IL; 2) Earth and Life Science Institute, Université catholique de Louvain, Louvain, Belgium; 3) Institute of Sciences of Food Production,National Research Council, Bari, Italy; 4) Plant Research International B.V., Wageningen, The Netherlands.In fungi, genes required for synthesis of secondary metabolites are often clustered. The FUM gene cluster is required for synthesis of fumonisins, a familyof toxic secondary metabolites produced predominantly by species in the Fusarium (Gibberella) fujikuroi species complex (FFSC). Fumonisins are a healthand agricultural concern because their consumption is epidemiologically associated with multiple diseases in humans and other animals. Among FFSCspecies, the FUM cluster is discontinuously distributed but uniform in gene order and orientation. In this study, we demonstrate that the FUM clusterexists in at least four different genomic contexts within the FFSC and that phylogenetic relationships derived from analyses of FUM cluster genes arecorrelated with genomic context, but are inconsistent with species relationships inferred from analyses of primary-metabolism genes. In addition, analysesof synonymous site divergence suggested that FUM cluster divergence predated divergence of the FFSC. These results are not consistent with transspeciesevolution of ancestral cluster alleles or with interspecies hybridization, but suggest duplication of the cluster within an FFSC ancestor andsubsequent loss and sorting of paralogous clusters in a manner consistent with the birth-and-death model of evolution previously described for multigenefamilies. A model based on horizontal gene transfer (HGT) could also explain these observations, but seems unlikely because it requires independenttransfer events from multiple unknown donors to multiple FFSC recipients. However, analyses of phylogenetic relationships and synonymous sitedivergence provided strong evidence that F. oxysporum strain FRC O-1890 acquired the FUM cluster via a relatively recent HGT event from F. bulbicola or aclosely related species within the FFSC. These results indicate that, as with other secondary metabolite clusters, species phylogenies do not provide anadequate picture of the complex evolutionary history of the FUM cluster within Fusarium.671. Chemotype predominance in Fusarium graminearum is not directly affected by the use of the fungicides trifloxystrobin and isopyrazam. MatiasPasquali, Tiphaine Dubos, Friederike Pogoda, Lucien Hoffmann, Marco Beyer. Environment and Agro-biotechnologies Department, CRP GABRIELLIPPMANN, Belvaux, Luxembourg.Mitochondrial respiration inhibitors are effective fungicides used to control wheat diseases in Europe. Since the beginning of the monitoring ofchemotype diversity in wheat fields in Luxembourg, we are trying to identify factors involved in the shift of chemotype prevalence (nivalenol vs. 15ADONvs. 3ADON). In this study we investigated whether the use of fungicides belonging to the respiratory inhibitors complex II and III (that are used in wheatfields for treating other diseases) may play a role in selecting a specific F. graminearum chemotype. Strains from Luxembourg and from a world collectionwith isolation dates ranging from 1969 to 2011 were chemotyped by genetic means and then analysed for their sensitivity to trifloxystrobin (inhibitor ofrespiratory complex III) and isopyrazam (inhibitor of respiratory complex II) using a microplate in vitro test on conidia. The maximum level of inhibitionwhich could be obtained by trifloxystrobin ranged from 14 to 65% for the 55 strains analyzed with no complete inhibition up to a concentration of 3mM.Fortyone isolates tested for their sensitivity towards isopyrazam were insensitive with the average rate of inhibition converging towards 28%. For bothfungicides, EC50 values did not significantly depend on the chemotype, suggesting that these two fungicides do not exert a direct pressure on the selection286