Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSpoint and are not able to follow dynamic interactions. Herein, the development of a microfluidic platform, to study the interplay of individual hyphae withbacteria in a confined compartment, is detailed. A microfluidic device, comprised of single, interconnected microchannels, is filled with medium. Thedevice is manufactured from the polymer, polydimethylsiloxane, which is bonded to a glass layer. Importantly, this polymer possesses some desirablecharacteristics, making it compatible with experiments of biological nature, for example it is oxygen permeable, and allows optical detection from 240 nmto 1100 nm. The fungus can easily grow into the microchannels from an agar plug that is placed next to a lateral opening. Subsequently, the device can beco-inoculated with bacteria through a separate inlet, allwoing changes in morphology, growth rate, and interaction patterns of the same hyphae upon theaddition of bacteria to be investigated over time. As an example, the interaction of the basidiomycetous model organism, Coprinopsis cinerea, and thegram-positive bacterium, Bacillus subtilis, which are both found in dung of herbivores, has been studied. Using these microfluidic platforms, it has beenobserved that B. subtilis cells attach to hyphae in an end-on manner. The frequency of attachment within hyphae and between different hyphae varies.Furthermore, the tips of the hyphae are not colonised by the bacterial cells. In summary, this approach can be used to study phenotypic changes ofbacteria and fungi over specific time periods. In future, it is envisaged that this data will be combined with fungal and bacterial genetic approaches.707. WITHDRAWN708. The French Fusarium Collection: a living resource for mycotoxin research. L. Pinson-Gadais, M. Foulongne-Oriol, N. Ponts, C. Barreau, F. Richard-Forget. INRA, UR1264-MycSA, 71 avenue Edouard Bourlaux, F-33883 Villenave d’Ornon, France.Fusaria are responsible for prejudicial diseases on cereal crops worldwide, such as crown rot and Fusarium head blight. Beyond economic losses due toinfection symptoms, these pathogens can produce several types of mycotoxins that are harmful to livestock and humans. They are extremely diverse atthe intra-specific levels in terms of types as well as quantities of toxins that a strain can produce. Developing appropriate strategies to limit contaminationwith Fusarium mycotoxins requires a greater knowledge about this variability. We have collected a large number of toxinogenic Fusarium strains. Ourassortment now includes about 800 strains, mostly from the species graminearum, culmorum, verticilloides, proliferatum, and temperatum. Species wereidentified based on morphology and real-time PCR. More than half of our strains were further characterized for toxin production using biochemical and/orreal-time PCR-based tools. We isolated about 70 F. graminearum strains from either wheat or maize grains originating from different French cerealproduction areas. Our results show a high representation of 15-acetyldeoxynivalenol-producing strains in our French samples. Within the samechemotype, we observe a large variability in toxin production levels. The F. graminearum strains were characterized with microsatellite markers and showa large genetic diversity. Two groups were delineated according to their genetic background, roughly corresponding to strains isolated from Europe in onehand and America in the other hand. Our results are also in agreement with the fact that only F. graminearum sensu stricto strains seem to be detected inFrance so far. The demonstrated genetic and phenotypic diversity provides a sound ground for countless downstream studies such as genetic associationand quantitative genetics to understand the determinism of toxin production. Such information should be doubtlessly considered in plant breeding effortsand other disease management strategies aimed at reducing the mycotoxin risk in food and feeds. Our collection is a valuable tool to improve ourunderstanding of toxigenic diversity in Fusarium species. It is managed through a database gathering all information collected on each strain, alreadyavailable upon request and soon publically available as a web-based interface.709. Chemical genetics: Discovery of novel fungicides and their targets in the phytopathogen Fusarium graminearum. G. Subramaniam, C. Mogg.Agriculture Canada, Ottawa, ON, Canada.Chemical genetics screen is based on the ability of small chemical molecules to bind to biological molecules and alter their function. Screening ofpharmaceutical libraries has revealed novel molecules effective against cancer and other diseases. We have adopted similar approach and identify bioactivecompounds that will block the growth and development of F. graminearum. We have developed a 96-well format to monitor the growth of F.graminearum in liquid media. The fungus is tagged with a green fluorescent protein (GFP) and the growth is monitored by the measurement offluorescence of the GFP. This format facilitates high throughput screening for small molecules that could potentially disrupt the growth of the fungus. Asproof of concept, we screened ~560 compounds from the TimTec NDL-3000 natural product collection (TimTec LLC, Newark, DE, USA) and identifiedseveral compounds with anti-Fusarium properties. One compound identified form our screen, “Antofine” was purified from Vincetoxicum rossicum andwas used in subsequent studies, to identify targets in the fungus. We used the gene deletion library of the budding yeast Sacchromyces cerevisiae toidentify targets for Antofine. GeneMANIA (http://www.genemania.org), an online multiple association network integration algorithm was used to uncoverinformation pertaining to genetic and physical interactions of these targets. Our efforts to identify targets in Fusarium against Antofine will be discussed.710. Functional characterization of an Aspergillus flavus polyketide synthase gene necessary for the synthesis of a sclerotium-specific pigment. J.W.Cary 1 , P. Harris-Coward 1 , K.C. Ehrlich 1 , P. Dowd 2 , S. Shantappa 3 , A.M. Calvo 3 . 1) US Department of Agriculture, ARS-SRRC, New Orleans, LA; 2) USDepartment of Agriculture, ARS-NCAUR, Peoria, IL; 3) Northern Illinois University, DeKalb, IL.The filamentous fungus, Aspergillus flavus, produces the toxic and carcinogenic, polyketide-derived family of secondary metabolites termed aflatoxins(AFs). In addition to the AF biosynthetic gene cluster, analysis of the A. flavus genome has identified 55 gene clusters predicted to be associated withsecondary metabolism. To date, very few of the metabolites produced by these clusters have been identified. Secondary metabolism is controlled byglobal regulators such as LaeA and VeA. In a veA knockout mutant we identified a significantly down-regulated polyketide synthase (PKS) gene belongingto cluster 27. Although the metabolite produced by this cluster was unknown, in silico cluster analysis predicted that cluster 27 would consist of the PKSgene and four other genes. qRT-PCR analysis confirmed that expression of the cluster 27 PKS (pks27) gene was down-regulated in the veA mutant.Inactivation of the pks27 gene resulted in loss of the dark pigment associated with A. flavus sclerotia. Sclerotia are survival structures produced bycondensation of mycelia and function as propagules in the field. Conidial pigmentation did not appear to be affected in the pks27 knockout strain. TLC andHPLC analysis of sclerotial extracts identified the cluster 27 metabolite as asparasone A. Insect feeding studies using wild-type and mutant sclerotiaindicated that the pigment may be acting as a feeding deterrent. To our knowledge this is the first report on the identification of a gene that encodes asclerotium-specific pigment. The pigment likely plays a role in sclerotial resistance to insect feeding and possibly other environmental stresses.711. Functional Analysis of the Pleurotus ostreatus Manganese-Peroxidase Gene Family. Tomer Salame, Doriv Knop, Dana Levinson, Oded Yarden,Yitzhak Hadar. Microbiology and Plat Pathology, Hebrew Unversity, Rehovot, Israel.Mn amendment to P. ostreatus cultures enhances degradation of recalcitrant aromatic compounds. Manganese peroxidase (MnP) isoenzymes are keyplayers in these processes. The MnP gene family is comprised of five Mn -dependent peroxidases (mnp3, 6, 7, 8 and 9) and four versatile-peroxidases(mnp1, 2, 4 and 5; VPs). In liquid medium, Mn amendment resulted in a drastic up-regulation of the predominantly expressed mnp3 and mnp9, and downregulationof mnp4. To obtain direct evidence for the role of these enzymes, we produced genetically-modified (knockout, knockdown and/or over-27th Fungal Genetics Conference | 295