necrotrophic effectors produced by the pathogen. Improved understanding of *P. nodorum*-wheat interactions is an effective way to address yield losses. The previous reference genome for *P.nodorum* was a fragmented assembly which prevented the efficient study of genes, their placement and genome evolution mechanisms. Resequencing of the SN15 strain with the Pacific Biosciences sequencing platform (PacBio) allowed assembly into 36 contigs. Contigs were placed onto a ~38.5 Mbp whole-genome restriction map (OpGen), resulting in 23 chromosomes comprising 37.89 Mbp of assembled sequence with an N50 of ~1.6 Mbp and L50 of 9.

Annotation of coding genes has been updated and manually curated, incorporating new RNAseq data from both *in vitro* and *in planta* experiments and applying new fungal-specific gene and effector prediction softwares. The resulting gene content was improved.

Comparisons between SN15, other isolates of *P. nodorum* and the closely related *P. avenaria* show that the smallest (~441Kbp) chromosome exhibits presence-absence variation (PAV) suggesting conditionally dispensable behaviour. The size and PAV patterns are consistent with a previously reported wheat/barley-specific chromosome suggesting that chromosome 23 may potentially harbour host-specific pathogenicity genes or play a role in driving genome plasticity and gene adaptation. We believe this improved genome assembly and the related analyses will assist effector discovery and illuminate new evolutionary studies across the Dothideomycetes.

**294F JGI fungal single cell genomics pipeline.** *Doina Ciobanu*¹, *Alicia Clum*¹, *Asaf Salamov*¹, *Wiliam Andreopoulos*¹, *Steven Ahrendt*¹,², *Alisha Quandt*³, *Igor Grigoriev*¹,², *Timothy James*³, *Jan-Fang Cheng*¹ 1) Genomics, Joint Genome Institute, Walnut Creek, CA; 2) Plant and Microbial Biology, University of California, Berkeley, CA, 94720, USA; 3) Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI, 48109, USA.

We developed a single cell genomics pipeline for noncultured fungal species and used it for sequencing seven early diverging fungi: *Blyttiomyces helicus* [Chytridiomycota], *Caulochytrium protostelioides* [Chytridiomycota], *Rozella allomycis* [Cryptomycota], *Syncephalis pseudoplumigaleata* [Zoopagomycotina], *Thamnocephalis sphaerospora* [Zoopagomycotina], *Piptocephalis cylindrospora* [Zoopagomycotina], *Dimargaris cristalligena* [Kickxellomycotina], and one Dikarya: *Metschnikowia bicuspidata* [Ascomycota]. The established pipeline for fungal single-cell genome recovery consists of seven steps: environmental sample collection and analysis, single cell isolation, single cell lysis and whole genome amplification, single cell amplified genome (SCAG) quality screening, best SCAG genome deep sequencing and assembly and finally best SCAG or combined SCAG (C-SCAG) annotation and functional analysis. Each of these steps can have an impact on the quality of the resulting genome and annotation. We validated each step of the pipeline using a set of quality check criteria such as start genome amplification (SGA), fold genome amplification (FGA), random twenty-mer uniqueness (RTU), genome GC content, assembled genome size plus a set of metrics for genome assembly quality and evaluated each using criteria for its predictability power in relationship to genome completeness, which was estimated using CEGMA (CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. G. Parra, K.Bradnam, I.Korf. Bioinformatics, V23, Iss 9, pp1061-1067). We examined all the factors that have a critical impact for fungal single cell genome recovery, the most important of which is amplification bias, which can be characterized by the combination of SGA, FGA, RTU and genome GC content. We offer a suite of protocols for a large scale workflow that can be applied to a wider range of organisms than fungi.

**295W SMRT sequencing reveals a transferrable pathogenicity chromosome in *Fusarium oxysporum* f.sp. *radicis-cucumerinum*.** *Peter van Dam*¹, *Like Fokkens*¹, *Michelle van der Gragt*¹, *Anneliek ter Horst*¹, *Balázs Brankovics*², *Petra M. Houterman*¹, *Martijn Rep*¹ 1) Molecular Phytopathology, University of Amsterdam, Amsterdam, The Netherlands; 2) CBS Fungal Biodiversity Centre, Utrecht, The Netherlands.

The genome of *Fusarium oxysporum* (Fo) is typically divided into a set of eleven 'core' chromosomes, shared by most strains and responsible for housekeeping, and one or several 'accessory' chromosomes. In Fo f.sp. *lycopersici*, one of these chromosomes was shown to be horizontally transferrable and responsible for pathogenicity towards tomato. In *de novo* Illumina assemblies, these accessory chromosomes are dispersed over many contigs, making it impossible to identify how many accessory chromosomes are present in a strain. We sequenced a strain of Fo f.sp. *radicis-cucumerinum* (Forc) using PacBio SMRT sequencing. The majority of the core chromosomes was assembled into single contigs, and a chromosome that shows all the hallmarks of a pathogenicity chromosome comprised two contigs. Part of this chromosome is very rich in repeats and transposons and contains all identified candidate effector genes, including homologs of *SIX6*, *SIX9*, *SIX11* and *SIX13*.

Through individual knockout of several *SIX* homologs as well as Horizontal Chromosome Transfer (HCT) towards a non-pathogenic strain (Fo47), we were able to show that (a) *SIX6* is an important effector for this root rot pathogen and (b) that this chromosome is indeed a pathogenicity chromosome for cucurbit infection. Strains that received this chromosome into a Fo47 background are fully pathogenic, essentially changing Fo47 into a virulent root rot pathogen infecting multiple cucurbits. One strain obtained by means of HCT showed a duplication of the entire pathogenicity chromosome and is still pathogenic, while another shows partial duplications and is reduced in virulence towards cucumber and watermelon but not towards muskmelon.

These observations allow us to conclude that also non-wilt-inducing Fo pathogens like Forc rely on effector proteins for successful infection and that the Forc pathogenicity chromosome contains all the information necessary for causing root rot of cucurbits. Moreover, the fact that three out of nine HCT strains have undergone large-scale chromosome alterations shows the enormous plasticity of Fo genomes.

**296T The genome of Paramicrosporidium saccamoebae highlights alternate adaptations to obligate intracellular parasitism in Cryptomycota and Microsporidia evolution.** *A. Quandt*¹, *D. Beaudet*², *D. Corsaro*³, *J. Walochnik*⁴, *R. Michel*⁵, *N. Corradi*², *T. James*¹ 1) Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI; 2) Canadian Institute for Advanced Research, Department of Biology, University of Ottawa, ON, Canada; 3) CHLAREAS Chlamydia Research Association, Nancy, France; 4) Molecular Parasitology, Institute for Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Austria; 5) Laboratory of Medical Parasitology, Central Institute of the Federal Armed Forces Medical Services, Koblenz, Germany.

Phylogenomic analyses have suggested that a clade comprising eukaryotic parasites with the smallest known genomes, Microsporidia, and the phylum known primarily from environmental sequences, Rozellomycota, are at the base of the fungal phylogeny. However, the ecological and genetic similarities between these distant relatives remains unclear. To gain insights into the genome diversity and biology of rostellids and their close relatives, we compared genome data of Rozellomycota and Microsporidia with the newly acquired nuclear and mitochondrial genomes of *Paramicrosporidium saccamoebae*; an intranuclear parasite of amoebae. Our analyses demonstrate that
Microsporidia are nested within Rozellomycota, which forms a paraphyletic clade, and reveal both lineage-specific loss of genes and previously unknown biological similarities between *P. saccamoebae* and its rozellid relatives. We show that the loss of complex I of oxidative phosphorylation and mitochondrial reduction has occurred more than once in the evolution of Rozellomycota, and that aspects of primary metabolism such as purine biosynthesis and the ability to synthesize several amino acids have been lost in *P. saccamoebae* but offset by horizontally transferred permeases that may help in acquiring these from their hosts. The general pattern of genome size and content reduction during the evolution in Rozellomycota and Microsporidia shows consistent but independent losses of genes as the result of variation in parasitic strategies (host, subcellular localization) within the clade.

**297F** Comparative genomics of the *Aspergillus* section *Flavi*. A. Sato¹, K. Matsushima¹, K. Ito¹, T. Mituyama² 1) R&D, Kikkoman Corporation, Noda, JP; 2) Artificial Intelligence Research Centre, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan.

*Aspergillus* section *Flavi* includes industrial fungi; *A. sojae* (*As*), *A. oryzae* (*Ao*), as well as aflatoxigenic fungi; *A. parasiticus* (*Ap*) and *A. flavus* (*Af*). It is difficult to clarify the evolutionary relationships among these four species because these fungi exhibit considerable morphological and physiological variations which hinder ordinary phylogenetics analysis. In order to clarify the relationship of these fungi at single base resolution, we performed re-sequencing and re-assembling of *As* NBRC 4239 upon our previous sequencing and obtained high quality 39.4-Mb chromosomal sequences with a 29-kb mitochondrial genome. We conducted computational genome comparison for these fungi and found that *As* is more homologous to *Ap* than to *Ao* and *Af*. In order to identify major factors to explain differences of genomic sizes (*As* and *Ap* 39Mb, *Ao* 37.2Mb, *Af* 36.9Mb) among these species, we conducted multiple compuation analyses. We found that genomic duplication can not be one of the factors because we did not find blocks to infer that *As* and *Ap* gained extra DNA by genome duplication. We conducted computational gene prediction by using Scipio (http://www.webscipio.org/) for these genomes. Then we computed gene-wise similarities among the species. The averaged alignment score over all predicted genes between *As* and *Ap* is 0.93 while that between *As* and *Ao* is 0.85. These results suggest that these fungi can be classified into two groups. We compared *As* and *Ap* genomes in terms of genomic regions of putative gene clusters for secondary metabolites. We found that *As* is deficient in some backbone genes which are essential for the secondary metabolite synthesis. This result suggests that *As* has lost abilities to generate some mycotoxins in the course of evolution. We developed a database of *As* in order to share our genome analysis results with the research community. Our database provides *As* genome browsing feature using Zenbu browser software (http://fantom.gsc.riken.jp/zenbu/). We integrated our genome annotation results into a GenBank formatted file which is supplied to the Model SEED server (http://modelseed.org/) to generate a SBML formatted genome-scale model. They can be downloaded from our database server at https://genome.cbrc.jp/sojae/.

**298W** Genome comparisons reveal transposable elements played a role in the evolution of *Ceratocystis albifundus*. *Magriet A van der Nest*, Michael J Wingfield, Brenda D Wingfield Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa.

Comparative genomics provides a powerful tool to investigate the processes that underlie the biology of fungi over evolutionary time. Genome-based studies have, for example, shown that transposable elements (TEs) play important roles in the evolution of pathogens and their adaptation to new environments. Here, we investigated the possible role that TEs might have had in the evolution and biology of the aggressive tree pathogen *Ceratocystis albifundus*. The publicly available genome of *C. albifundus* was complemented with sequenced genomes of four additional isolates from a wide geographic range and on different hosts. The genomes were compared in terms of size, sequence similarity, gene content, and the pathways and processes that they encode. Finally, we used the REPET pipeline to identify and annotate the TEs present in five *C. albifundus* genomes. The results showed that these genomes were similar in genome size, gene content and overall sequence similarity. These similarities also extended to the pathways and processes encoded by each genome. The most obvious differences among the five isolates were evident in the distribution and number of repeated sequences, TEs, and/or proteins involved in TE activity. The presence and activity of these elements are commonly responsible for the accelerated evolution in the genomes of fungal pathogens and they most likely contributed significantly to the biology and evolution of *C. albifundus*.

**299T** Characterization and manipulation of sesquiterpene biosynthetic pathways in *Hypholoma* species. *Suhad Al-Salihi*, Andy Bailey, Gary Foster Molecular Plant Pathology and Fungal Biology Group, School of Biological Sciences, University of Bristol 24 Tyndall Ave, Bristol BS8 1TQ, UK.

The increase in microbial resistance to antibiotics highlights the need to intensify efforts to discover novel biological active compounds which have the ability to kill or inhibit resistant pathogenic bacteria. Despite the prominence of Basidiomycetes as bioactive compounds producers, only a few species have been studied in detail as a source of novel antibiotics. Both *Hypholoma fasciculare* (Hfas) and *Hypholoma sublateritium* (Hsub), are a prolific producers of pharmaceutically important sesquiterpenoids and triterpenoids, and their antimicrobial activity against *Bacillus subtilis*, *Escherichia coli*, *Staphylococcus aureus*, and *Saccharomyces service* has been confirmed in this work.

Two "foreign" genes (hygromycin resistant and green fluorescent protein) have been expressed heterologously via a developed ATMT method, and the use of the Hsub *gpd* promoter as an efficient regulator element is described for the first time in this research.
Both the in-house generated genome of Hfas and the published genome of Hsub, were mined for terpenoid core genes using antiSMASH and local blast search. This has revealed 17 and 16 terpene synthases from Hfas and Hsub respectively. Phylogenetic comparison with characterized basidiomycete terpene cyclases has categorized these into three different groups based on predicted of cyclization patterns, confirming that these two mushrooms are capable of producing a wide range of terpenoids. Further syntenic comparison of the two genomes carried out and six twinned gene clusters were characterized in both *Hypholoma*.
Chemical investigation so far has confirmed the production of the sesquiterpene compound "Naematolin" from both species. The structure of this compound has been confirmed via NMR analysis, and a further work is on-going to identify the gene cluster responsible for the production of this antimicrobial compound.
**300F De Novo genome sequencing and transcriptome analysis of two *Venturia inaequalis* strains.** Cristina Miguel Rojas¹, Brad Cavinder¹, Janna Beckerman², George Sundin³, Jeffrey P. Townsend⁴,⁵, Frances Trail¹,³ 1) Plant Biology, Michigan State University, East Lansing, MI; 2) Department of Botany and Plant Pathology, Purdue University, IN; 3) Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI; 4) Department of Biostatistics, Yale University, New Haven, CT; 5) Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT.

*Venturia inaequalis* is a Dothideomycete fungus that causes apple scab, one of the most devastating diseases of apple. Recently, *Venturia* has become a powerful model to study the pathogenesis of hemi-biotrophic fungal plant pathogens. We have performed *de novo* high-throughput whole-genome sequencing of two *Venturia inaequalis* strains featuring different virulence genes. To determine the genetic basis of the differences in virulence, we are comparing their genomes. To further identify infection-specific genes, we carried out a transcriptomic analysis of the spore germination process for one of the two strains during germination on a defined medium and during germination on apple leaves. The evolution of the transcriptome will additionally be analyzed in a set of species possessing divergent mechanisms for plant penetration.

**301W Whole genome DNA-methylation (methylome) profiling during heterokaryosis in the Agaricomycetes.** R.A. Powers, T.Y. James Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI.

DNA methylation of the 5' position of cytosines, a well-known form of 'epigenetic' modification, has been shown to be important in such diverse processes as the formation of human cancers, development in multicellular eukaryotes, and the silencing of transposons and repetitive elements in plants, animals, and fungi. Despite the importance and apparent conservation of DNA methylation across diverse clades of eukaryotes, we still lack a basic understanding of its roles in the mushroom-forming fungi of the Agaricomycetes. While it has been shown that DNA methylation in CpG (5' Cà G 3') contexts is important for the silencing of repetitive DNA elements in fungi, we lack a comprehensive understanding of DNA methylation at the whole genome level in the Basidiomycota. In particular, we know little of how whole genome DNA methylation ("methylome") patterning differs both during different life-stages within a species, as well as how methylomes differ between taxa. Using whole-genome bisulfite sequencing (WGBS), which involves the treatment of genomic DNA with sodium-bisulfite followed by whole genome next generation sequencing, we have generated genome-wide methylation profiles of two haploid homokaryotic isolates from five taxa across diverse clades of the Agaricomycetes: *Coprinopsis cinerea*, *Heterobasidion irregulare*, *Wolfiporia cocos*, *Coprinellus disseminatus* and *Cyathus stercoreus*. Here, we report that the amount of genomic methylation of cytosines in CpG contexts is species specific and ranges from of a low of ~2% in *Heterobasidion irregulare* to ~12.5% in *Cyathus stercoreus*. Further, we show that there is a high degree of variation in CpG methylation percentages between haploid strains of the same species for most of the species tested. Lastly, we examined the role of changes in methylome patterning during mating and heterokaryosis and show that DNA methylation patterns change during the transition for haploid to heterokaryon, and that these changes are also species specific.

**302T Applications of the genome sequence of *Cercospora zeina*, causal agent of gray leaf spot disease of maize.** Dave Berger¹, Nicholas Olivier¹, Velushka Swart¹, Yao-Cheng Lin², Bridget Crampton¹, Irene Barnes³, Yves Van de Peer²,⁴, Pedro Crous⁵, Burton Bluhm⁶ 1) Department of Plant and Soil Sciences, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa; 2) VIB Department of Plant Systems Biology, Department of Plant Biotechnology and Bioinformatics, Ghent University, Belgium: 3) Department of Genetics, FABI, University of Pretoria, South Africa; 4) Genomics Research Institute, University of Pretoria, South Africa 5) CBS Fungal Biodiversity Centre, Utrecht, The Netherlands; 5) Department of Plant Pathology, University of Arkansas, Fayetteville, USA; 6) CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands.

*Cercospora zeina* is a Dothideomycete fungus with a lifestyle adapted to invasion and multiplication in maize leaves. Symptoms on susceptible maize leaves are characterized by grayish matchstick-like lesions parallel to leaf veins, hence the name gray leaf spot (GLS) disease. Although *C. zeae-maydis* has been better studied as agent of GLS in the USA, *C. zeina* is the causal agent of GLS in Africa. We sequenced the genome of an African isolate of *C. zeina*, and will illustrate three applications from these data. First, the sequence was mined for microsatellite markers, which were used for a population genetics study of the pathogen from commercial maize in South Africa. We observed a diverse panmictic population across the country indicating widespread dispersal. Second, the cercosporin biosynthetic pathway was annotated, revealing pseudogenization of an oxido-reductase gene, providing an explanation for lack of cercosporin production by this species. Third, we are developing a bioinformatics pipeline to identify single copy syntenic genes across sequenced Dothideomycetes that can be used to resolve unidentified species within the genus *Cercospora*.

**303F Whole-genome haplotype sequencing of Puccinia coronata f. sp. avenae, causal agent of oat crown rust disease.** M. Miller¹,², V. Omidvar¹, Y. Zhang¹, B. Schwessinger⁶, J. Rathjen⁶, C. Hirsch¹, C. Raley⁵, R. Park³, P. Dodds¹,⁴, S. Kianian¹,², M. Figueroa¹ 1) University of Minnesota, MN, USA; 2) USDA-ARS Cereal Disease Laboratory, MN, USA; 3) University of Sydney, NSW, Australia; 4) CSIRO Agriculture and Food, ACT, Australia; 5) Leidos Biomedical Research, MD, USA; 6) Australian National University, ACT, Australia.

Oat crown rust caused by *Puccinia coronata* f. sp. *avenae* (*Pca*) is one of the most destructive pathogens affecting grain production. *Pca* shows high genetic variability and resistance genes deployed in the field are often overcome in just a few years, which is of particular importance given that domestic oat production cannot keep pace with U.S. demand. The genomic resources necessary for a greater understanding of molecular mechanisms of virulence and more targeted resistance deployment strategies of *Pca* are underdeveloped. We generated *de novo* genome assemblies of two *Pca* isolates with contrasting virulence phenotypes (races LBBB and STTG). Genomic analyses of rust fungi are complicated by both the dikaryotic nature in the most readily accessible life stages and also the high percentage of repetitive elements present in the genome. To overcome these obstacles, we implemented emerging technologies to assemble haplotigs of urediniospores to produce a high quality reference genome. Long-read single-molecule PacBio and short-read Illumina sequences were analyzed in parallel to accurately phase the genome using FALCON and FALCON-Unzip genome assembly algorithms. The high quality genome references were annotated utilizing expression data from a variety of tissues and life stages, including haustoria isolated from infected tissues, and a catalog of predicted effectors was generated. Moreover, we compared sequence variation and repetitive element repertoires to assess the variability between nuclei and inter-isolate comparisons were used to characterize the genomic landscape of this fungal species. We are also embarking on a resequencing project of 60 isolates to describe the pan-genome of *Pca* and variability among
isolates. Ultimately, this project will provide genomic resources to study heterozygosity, conserved and variable genomic regions, and ultimately the evolution of pathogenicity mechanisms in *Pca*.

**304W Penicillium genomics to solve postharvest apple decay.** Jiujiang Yu¹, Guangxi Wu², Guohua Yin³, Hui Peng⁴, Verneta Guskins¹, Wayne, W. Jurick II¹, Joan, W. Bennett³ 1) USDA/ARS, Beltsville Agricultural Research Center, Beltsville, MD; 2) Colorado State University, Fort Collins, CO; 3) Rutgers University, New Brunswick, NJ; 4) Pennsylvania State University, University Park, PA.

Post-harvest decay of apple and pears is caused by Penicillium species, mainly *P. expansum*. It is commonly called the Blue mold. The highly virulent species *P. expansum* (R19) is the major fungal species that causes blue mold. On the contrary, *P. solitum* (RS1) is the weakly virulent species we identified. Genome-wide analysis of the genetic components and their regulatory mechanisms may provide insight into their virulence for devising pathogen-specific control strategies. For that reason, the two species were sequenced and analyzed. The genome similarities and differences were examined. A total of 10,560 and 10,672 protein coding sequences were identified and contained 41 and 43 unique genes in *P. expansum* (R19) and *P. solitum* (RS1), respectively. The genes responsible for infection in apple decay could be among those unique genes identified in the virulent strain *P. expansum* (R19). Further investigations to characterize their specific function of these genes are underway through gene knock-out experiments.

**305T A haplotype phased reference genome and a transformation system for the wheat stripe rust fungus *Puccinia striiformis* f. sp. *tritici*.** B. Schwessinger¹, Y. Hu¹, W. Cuddy²,³, R. Park², J. Rathjen¹ 1) Research School of Biology, The Australian National University, Acton ACT 2601, Australia; 2) Department of Plant and Food Sciences, Faculty of Agriculture and Environment, University of Sydney, Plant Breeding Institute, Private Bag 4011, Narellan, NSW, 2567, Australia; 3) Elizabeth Macarthur Agricultural Institute, NSW Department of Primary Industries, Woodbridge Road, Menangle, NSW, 2568, Australia.

Stripe rust of wheat, caused by the obligate biotrophic fungus *Puccinia striiformis* f. sp. *tritici* (*Pst*), is a major threat to wheat production world-wide with an estimated yearly loss of US$ 1 billion. *Pst* undergoes a complex multi-spore life-cycle and infects wheat during its asexual stage mostly via reinfection by dikaryotic urediniospores. The highly heterozygous genome together with its obligate biotrophic life-style has hindered progress in understanding the molecular and genetic mechanisms of *Pst* pathogenesis and evolution. Current genome assemblies are highly fragmented and unphased making downstream analysis at a whole genome level challenging. Here we present a 80% haplotype phase reference genome for one Australian *Pst* founder race based on single molecule long read sequencing technologies. We will explore differences between the two haplotypes in regards to nucleotide and structural variations, transposable element and gene content. In addition, we will present a new Agrobacterium based *in vitro* transformation protocol for *Pst*, which might be applicable to rusts in general.

**306F Molecular Characterization of Horizontally Transferred Genes in a Zygomycete Fungus *Choanephora cucurbitarum*.** B. Min, H. Park, J. Byun, H. Shin, I. Choi Korea University, Seoul, KR.

A zygomycete fungus, *Choanephora cucurbitarum* is a causal agent for blossom rot in cucurbits and other plants. We recently reported the genome sequence of *Choanephora cucurbitarum* KUS-F28377 isolated from a Korean squash. The genome was assembled to a size of 29.1 Mbp and 11,977 protein-coding genes. Comparing to ten Mucorales and two Glomerales genomes, we identified 1,326 orphan genes only found in the genome. One orphan gene family had 31 members presenting F-box domain. It is interesting that the genome contained 11 Streptomyces subtilisin inhibitor-like domains (SSIDs). SSID has been found only in bacterial genomes mainly in *Streptomyces* species. SSIDs are known to be related to defense against plant immunity. We cloned and characterized the molecular function of the SSID-containing genes in *E. coli*. Overall, these findings will help us to understand how zygomycetes are associated with plants.

**307W Trichoderma reesei complete genome sequence reveals chromosome architecture, repeat-induced point mutation and partitioning of CAZyme gene clusters.** Wan-Chen Li¹,²,³, Chien-Hao Huang³,⁴, Chia-Ling Chen³, Yu-Chien Chuang³, Shu-Yun Tung³, Ting-Fang Wang¹,³ 1) Taiwan International Graduate Program in Molecular and Cellular Biology, Academia Sinica. Taipei 115, Taiwan; 2) Institute of Life Sciences, National Defense Medical Center, Taipei 115, Taiwan; 3) Institute of Molecular Biology, Academia Sinica. Taipei 115, Taiwan; 4) Institute of Genome Sciences, National Yang-Mang University, Taipei 112, Taiwan.

*Trichoderma reesei* (Ascomycota, Pezizomycotina) QM6a is a model fungus for a broad spectrum of physiological phenomena, including plant cell wall degradation, industrial production of enzymes, light responses, conidiation, sexual development, polyketide biosynthesis and plant-fungal interactions. We integrated Pacific Biosciences and Illumina sequencing platforms for the highest-quality genome assembly yet achieved, revealing seven telomere-to-telomere chromosomes (34,922,528 bp) with 2009 newly-predicted genes and >1.5 Mb of new sequences. Most new sequences are located on AT-rich isochores, including 7 centromeres, 14 subtelomeres and 2349 interspersed AT-rich islands. The seven QM6a centromeres separately consist of 24 conserved repeats and 20 centromere-encoded genes. We demonstrate that sexual crossing readily induced cytosine-to-thymine point mutations on both tandem and unlinked duplicated sequences. Comparative bioinformatics analyses reveal that *Trichoderma reesei* has evolved a robust repeat-induced point mutation (RIP) system to accumulate AT-rich sequences, with longer AT-rich isochores having more RIP mutations. Our analyses, together with the complete genome sequence, provide a better blueprint for biotechnological and industrial applications.

**308T Genomics of the first 100 Aspergilli.** A. Kuo¹, R. Riley¹, S. Mondo¹, S. Haridas¹, A. Salamov¹, F. Korzeniewski¹, B. Simmons², S. Baker², M. Andersen³, I. Grigoriev¹ 1) DOE Joint Genome Institute, Walnut Creek, CA; 2) DOE Joint BioEnergy Institute, Emeryville, CA; 3) Technical University of Denmark, Kongens Lyngby, Denmark.

*Aspergillus* is a ubiquitous and phenotypically diverse genus of filamentous Ascomycota, many of which play key roles as fermenters in food production, platforms for biotechnology and industrial production of enzymes and chemicals, plant and opportunistic animal pathogens, and agents of agricultural toxigenesis and biomass conversion for bioenergy. As part of a Joint BioEnergy Institute initiative to characterize the entire genus, the Joint Genome Institute will sequence, assemble, and annotate the genomes of each of the ~300 species of the genus *Aspergillus*. To accomplish this massive task in a timely manner without sacrificing quality, we streamlined and optimized our processes for *Aspergillus* genomes. Over the past year we have released on MycoCosm the genomes of 100 *Aspergillus* sp. which represent a broad spectrum of phylogenetic diversity and gene content, including significant variability of transporters, carbohydrate-active enzymes,
proteases, and secondary metabolism clusters. The high resolution of genomic differences between closely related species is being mapped to their distinctive phenotypes to improve gene annotation in the entire genus. The next 100 species are expected soon.

**309F Comparative genomics of *Aspergillus flavus* S and L morphotypes provides insights into niche adaptation.** Mana Ohkura, Peter Cotty, Marc Orbach School of Plant Sciences, University of Arizona, Tucson, AZ.

*Aspergillus flavus* is a widely distributed saprotrophic fungus and the most common causal agent of aflatoxin contamination on food crops. Despite the notoriety of *A. flavus* to produce aflatoxin, strains vary greatly in their levels of toxin production and atoxigenic strains are not uncommon in nature. Within *Aspergillus flavus*, there are two distinct morphologies, namely morphotypes: the S morphotype produces numerous small sclerotia and limited conidiospores and the L morphotype produces relatively fewer large sclerotia and abundant conidiospores. S strains are consistently toxigenic, whereas L strains vary greatly in toxin production ranging from atoxigenic to highly toxigenic. Morphological differences between the two morphotypes suggest the production of abundant sclerotia in S strains is advantageous for long-term survival in the soil, whereas the production of abundant conidia in the L strains is advantageous for aerial dispersal to the phyllosphere. The selection pressure to maintain aflatoxin production may be higher in the soil compared to the phyllosphere, leading to the retention of consistently toxigenic S strains in the soil. In contrast, the selection pressure may be lower in the phyllosphere, allowing the survival of atoxigenic or low toxin-producing L strains in that niche. To develop hypotheses on differential niche adaption, we compared the genomes of three S strains and three L strains to identify structural differences and genes unique to each morphotype. A 530 kb inversion was identified between the morphotypes that affects a secondary metabolite gene cluster and a cutinase gene on the margins. The L strain genomes contained 10 deletions, many of which involve secondary metabolite genes. Each morphotype had unique genes that play a role in carbon/nitrogen metabolism, secondary metabolism, and antimicrobial defense. These findings indicate that the genomes of the two morphotypes differ beyond developmental genes, and that they may have diverged as they adapted to their respective niches.

**310W Are G protein-coupled receptor proteins involved in thigmoregulation of aflatoxin inhibition by *Aspergillus flavus*?** R.R. Sweany, K.E. Damann, Jr. Plant Pathology and Crop Phys, Louisiana State University, Baton Rouge, LA.

*Aspergillus flavus* can contaminate corn, groundnuts and other oil seed crops with acutely toxic and carcinogenic aflatoxin. Atoxigenic biocontrol strains of *A. flavus* reduce aflatoxin of toxigenic strains in a thigmoregulated manner. G protein-coupled receptor (GPCR) mutants of *A. flavus* were obtained from Nancy Keller at the University of Wisconsin. Since GPCRs are membrane bound proteins involved in signaling, we determined whether loss of signal receptor proteins affected intraspecific aflatoxin inhibition. Aflatoxin production was quantified using HPLC of extracts from four-day old, glucose-salts medium cultures grown in 24-well plates. Several atoxigenic isolates, both commercially developed biocontrol strains and isolates from Louisiana, were screened against the wild-type CA14 and the CA14N1 *Nit* mutant (the genetic background for gene knock outs) for intraspecific aflatoxin inhibition. Only two strains from Louisiana completely inhibited aflatoxin production of CA14 and CA14N1. One inhibitory biocontrol strain was grown with the GPCR mutants to look for loss of biocontrol function. The biocontrol strain inhibited aflatoxin production in all mutants. Toxigenic strains can also have biocontrol ability and inhibit aflatoxin production, therefore the mutants were grown with four toxigenic strains representing small or large sclerotial strains of both mating types. Only two GPCR mutants did not inhibit aflatoxin production of the toxigenic strains. When non-inhibitory GPCR mutants were germinated 12 hours prior to inoculation (due to slow growth) with competing toxigenic strains, the ability to inhibit toxin production was restored. Giving the slow-growing GPCR mutants a head start presumably reduced aflatoxin production because competing germinated hyphae now touched within the 1st 12 hours. This reiterates earlier observations that touch must occur early during the germination of the toxigenic isolate, otherwise aflatoxin production will be unaltered by a competing strain. Single G protein-coupled receptor protein knockouts did not change aflatoxin inhibition, but further studies are needed during the 12-18 hour critical window to understand what signaling leads to successful intraspecific aflatoxin inhibition.

**311T Centromere dynamics and the formation of dicenteric chromosomes in *Zymoseptoria*.** Klaas Schotanus¹,², Christoph J. Eschenbrenner¹,², Lanelle R. Connolly³, Mareike Moeller¹,², Kristina M. Smith³, Michael Freitag³, Eva H. Stukenbrock¹,² 1) Environmental Genomics, Christian-Albrechts University of Kiel, Kiel, Germany; 2) Max Planck Institute for Evolutionary Biology, Plön, Germany; 3) Department of Biochemistry and Biophysics, Oregon State University, Corvallis OR, USA.

Fungal plant pathogens in the genus *Zymoseptoria* have diverged only 12,000 years ago, and since then *Z. tritici* has specialized to infect wheat and become an important crop pathogen with worldwide distribution. In contrast, *Z. pseudotritici* and *Z. ardabiliae* occur on wild grasses at the center of origin in Iran.

All three species have two types of chromosomes, namely gene-rich core chromosomes enriched with euchromatic histone marks, and gene-poor accessory chromosomes enriched with heterochromatic histone marks. We previously showed that centromeres and telomeres of core and accessory chromosomes are indistinguishable in terms of sequence composition and structure, and demonstrated that centromeres of *Z. tritici* are small, are not enriched with DNA repeats and sometimes have actively transcribed genes abutting or within the short, 5-14 kb centromeric DNA sequences.

To address the evolution of centromeres we conducted a comparative analysis of centromeres in *Z. tritici*, *Z. pseudotritici* and *Z. ardabiliae*. The centromere specific histone H3 (CenH3) was tagged at the amino terminus with GFP in *Z. ardabiliae* and *Z. pseudotritici*, allowing us to identify centromeric sequences in the genome of the two species by ChIP-seq and mapping of Illumina reads to PacBio assemblies of *Z. ardabiliae* and *Z. pseudotritici*.

Overall, centromeres of *Z. pseudotritici* and *Z. ardabiliae* resembled those of *Z. tritici* as they are short (5-16 kb) and contain a low amount of repetitive DNA. However, compared to *Z. tritici*, five *Z. pseudotritici* centromeres were found in non-syntenic regions and lack homology between the species, a remarkable finding considering that we previously found that centromere position is conserved in syntenic regions of more divergent *Fusarium* species.

We also found two small dicentric chromosomes in *Z. pseudotritici*, though it remains to be seen whether both centromeres are functional. They are separated by 11 & 50 kb of interstitial regions that are enriched for transposable elements and the gene density left and right of the two centromeres differs. We hypothesize that the dicentric chromosomes could be the products of a fused chromosome with different gene densities. By comparative genomics, we identified homologous sequences of the dicentric chromosomes in 16 and 19 isolates
respectively, out of 27 total isolates sequenced, suggesting that the sequences of the dicentric chromosomes are conserved in the majority of the *Z. pseudotritici* isolates sequenced.

**312F Advances in chemical genetic tools and impact on the research and development of novel crop protection products.** Siân Deller, Helen Carter, Helen Blake, Eileen Scott, *Michael Csukai* Syngenta, Jealott's Hill, GB.

Chemical control agents are likely to remain a part of the mixture of control strategies open to farmers for the foreseeable future. In order to support the discovery and development of new active ingredients a wide range of chemical genetic tools are utilised and are under constant development. Historically many of these tools used the model organism *Saccharomyces cerevisiae*, but improvements in genomic information and tools make it possible to perform research directly in field-relevant plant pathogens. Some of the wide range of tools used in support of new fungicide discovery will be outlined, including:

- Forward genetic tools, which have long been the mainstay of mode of action diagnosis, provide an unbiased approach for the identification of the molecular target of a chemical inhibitor. If resistant mutants can be generated, the technique can now be applied to any pathogen, providing a reference genome is available and single nucleus cells/spores can be isolated.

- Libraries of engineered model organism strains. Chemistry with lower resistance risk is often associated with the inability to isolate resistant mutants in the plant pathogen of interest. In this case unbiased genetic tools are still of value and model organisms are employed. Haploinsufficiency or overexpression-induced resistance within a yeast library can provide information on proteins or protein families targeted by novel chemistry. Hypotheses can then be based on the mutations seen, and compared to information from additional phenotypes such as biochemical pathway inhibition and microscopic observations.

The improvements in transformation procedures, genetic engineering techniques and availability of multiple selectable markers make it possible to test hypotheses in fungal pathogens of economic importance such as *Zymoseptoria*. Additionally, genes from any species can be tested in a uniform host cell background, for instance, by heterologous expression. Furthermore, these tools can be used to assess the lifetime of a new fungicide product. Mutations induced in the lab or discovered in the field can be assessed for their importance in different genetic backgrounds and an assessment made of the impact on sensitivity and fitness. Advancing molecular biology tools and understanding of phytopathogens are helping to inform and support the development of ever safer and more effective chemical crop protection strategies. How these tools have been used to study Zymoseptoria will be shown.

**313W New Zealand: The Home of Hobbits and Fungicide Resistant Yellow Rust?** N.M. Cook¹, J. Maintz², S. Chng³, D.G.O. Saunders¹,² 1) Earlham Institute, Norwich, GB; 2) John Innes Centre, Norwich, GB; 3) Plant & Food Research, Christchurch, NZ.

Every year crops come under attack from cereal pathogens, which reduces their yield and the economic viability of growing crops. One major threat to wheat production worldwide is caused by yellow rust (*Puccinia striiformis* f. sp. *tritici*) which can lead to severe yield losses as 88% of wheat varieties are susceptible to yellow rust infection. This disease can be treated effectively by the use of fungicides but these are under threat by the potential for yellow rust to develop fungicide resistance. For instance, recently developed single site fungicides can easily be overcome by a single mutation within the fungicide target site. One such class of fungicides are the demethylation inhibitors (DMIs) which target the CYP51 gene involved in sterol biosynthesis.

In this study, we identified for the first time six isolates of yellow rust from New Zealand which contain a Y134F mutation within the CYP51 gene. This is homologous to the Y137F mutation previously characterised in many other fungal pathogens including *Zymoseptoria tritici* and is known to cause a reduction in sensitivity to DMIs. The yellow rust isolates containing this mutation were from two distinct genetic backgrounds suggesting the mutation arose twice independently in New Zealand. Although the Y134F mutation is heterozygous in these isolates, the mutated allele was shown to be preferentially expressed in all six isolates. Fungicide spray tests to determine if the Y134F mutation does indeed confer fungicide resistance for wheat yellow rust are currently underway.

**314T Transcriptome analyses of differentially expressed genes in the *Lentinula edodes*.** H Song¹, D Kim², J Kim¹ 1) Bio-Environmental Chemistry Dept, WKU, South Korea; 2) Molecular Biology Dept, JBNU, South Korea.

The edible mushroom *Lentinula edodes* has been utilized as a popular medicine, as well as food, because extracts from its mycelium and fruiting body have revealed several therapeutic properties. However, the deficit of *L. edodes* genomic information study has been barrier to the advancement of functional genomics research. High-throughput Illumina RNA-seq was used for deep gene analyses of the transcriptome of poly (A)+RNA from mycelium and fruiting body in *L. edodes*. About 141.77 million reads were obtained, trimmed, and de novo assembled into 32,001 transcripts. Based on sequence orientations determined by a BLASTX search against the NR fungi, Uniprotkb fungi, COG, GO, and KEGG databases, 27,569 (86.15%) contigs were assigned putative descriptions. When the expression profiles of mycelium and fruiting body were compared, 2,898 DEGs were detected, including 880 upregulated and 2,018 downregulated. Among the 27 COG categories, the cluster for general function prediction represents the largest group followed by 'Secondary metabolites biosynthesis, transport, and catabolism'. Results of GO analysis, DEGs were involved in diverse biological process. The most highly enriched GO category was cytoplasm, intracellular membrane-bounded organelle, and protein metabolic process. Analysis of 19 KEGG categories revealed that the DECs were significantly enriched in metabolic and signal transduction pathway. The pathways with most representation by the unique sequences were translation and carbohydrate metabolism.

**315F Comparative genomics of lichen mycobionts.** A. Kuo¹, D. Culley², O. Mueller³, P. Dyer⁴, J. Magnuson², F. Lutzoni³, I. Grigoriev¹ 1) DOE Joint Genome Institute, Walnut Creek, CA; 2) Pacific Northwest National Laboratory, Richland, WA; 3) Duke University, Durham, NC; 4) University of Nottingham, Nottingham, UK.

Lichens are mutualistic symbioses usually between an alga (the photobiont) and a fungus (the mycobiont). The photobiont contributes fixed carbon to the partnership, while the mycobiont provides a physical structure that encloses the photobiont, roots the partners to a substrate, and acquires nutrients often of air-borne origin. Lichens are an ancient (possibly 600 Mya) and highly successful adaptation, covering 8% of terrestrial ecosystems, and comprising as much as 20% of fungal species diversity. The lichen lifestyle has evolved multiple times and occurs in at least 5 fungal classes. As part of an ongoing effort to represent the full phylogenetic and ecological diversity of fungi in the Joint Genome Institute MycoCosm and the 1000 Fungal Genomes Project, we have partnered with members of the lichen research community to sequence, assemble, and annotate several lichen mycobionts and photobionts. Initial comparison of 5 publicly
available mycobiont genomes from 3 distinct lichen-forming clades with their nearest non-lichenizing relatives has revealed varied combinations of secondary metabolite synthase, transporter, and protease gains and losses. The diversity of gains and losses suggest that the independent evolutionary transitions to the lichen lifestyle may have involved different gene-level adaptations. We expect to expand on these findings by examining additional functional characters, and by sequencing more genomes, as well as importing more genomes from our partners and from the public domain.

**316W Comparative mitochondrial genomics of nine *Malassezia* species reveals diverse mitochondrial genome organization and evidence of homologous recombination between large inverted repeats.** *C. D. Baudo*¹, *E. Westhus*², *J. C. Kennell*¹ 1) Department of Biology, Saint Louis University, Saint Louis, MO; 2) Center for Outcomes Research, Saint Louis University, Saint Louis, MO.

*Malassezia* yeast species occupy broad ecological niches from deep-sea vents to the skin of animals where they are associated with a number of chronic skin diseases. Mitochondrial (mt) haplotypes have been proposed to be associated with virulence in the closely related fungus *Cryptococcus gattii* highlighting the need to characterize mt genomes of other fungal pathogens. In this report, the mt genome of 19 isolates from nine *Malassezia* species were assembled, annotated, and compared. Four of the nine species, *M. sympodialis*, *M. restricta*, *M. globosa*, and *M. pachydermatis* share identical gene order and form a distinct branch on a maximum likelihood phylogenetic tree. A distinguishing feature detected in all but one species, *M. yamatoensis*, is the presence of a large inverted repeat (LIR) ranging from 3.5-8.7 kb within the mt genome. Closely related species have a higher percent nucleotide identity in the LIR than the intergenic regions, yet this relationship reverses as evolutionary distance increases between the species. Substitution rate (Ks) analysis of a protein coding gene (PCG) in the LIR, *atp9*, was found to have lower Ks than PCGs in the single copy region. These data suggest relaxed selection pressures exist on noncoding sequences in the LIR compared to the intergenic regions and PCGs are preserved through copy correction. The *M. sympodialis* ATCC 42132 mt genome was also assembled from single molecule, real-time (SMRT) reads and inspection of reads extending from the LIR into the inter-LIR region found the LIR region present in multiple orientations. The analysis indicates homologous recombination occurs between repeat regions that inverts the inter-LIR region producing variable genomic configurations. The comparative genomic analysis presented here provides valuable insight into the evolution of mt genomes and the role of homologous recombination in generating novel genomic arrangements.

## Fungal Diversity

**317T How accurate biological source information enables data verification and reuse.** *B. Robbertse*¹, *P. Strope*¹, *R. Gazis*², *P. Chaverri*³,⁴, *C. Schoch*¹ 1) National Center for Biotechnology Information, National Library of Medicine, NIH, Bethesda, MD; 2) University of Tennessee, Department of Entomology and Plant Pathology, Knoxville, TN; 3) University of Maryland, Department of Plant Science and Landscape Architecture, College Park, MD; 4) Escuela de Biología, Universidad de Costa Rica, San Pedro, San José, Costa Rica.

The ITS (internal transcribed spacer) RefSeq project (NCBI BioProject accession PRJNA177353) aims to provide accurate organism name to sequence associations and additional information (public specimen sources) to confirm this association by independent evaluation. Since curators at NCBI have started collecting and annotating type material (identifiers that ties formal biological names to physical specimens), the most immediate impact has been improving the accurate identification of genome and barcode data. Unfortunately, the primary sequence records from type material frequently contain only the minimum information with no other metadata beyond strain or isolate identifiers. Although the RefSeq ITS project does add public collection identifiers and verify the specimen-sequence-name triangle, no additional source data is added on a regular basis. Gathering source information from protologues (species descriptions) scattered among several journals, most of which are not easily accessible, is a high curation burden. However, when researchers in the community get involved, it becomes a more feasible task. As an example, a thorough curation of one of the top ranking important fungal genera, namely Trichoderma was recently completed. The effort resulted in several verified data associations beyond name-sequence associations (eg: isolation source, location etc.). We present the outcome of this process and highlight the importance of source information.

**318F Molecular analysis of the dust mycobiome from homes of patients with aspergillosis.** *Ahlam Alanazi*¹, *Paul Bowyer*², *Malcolm Richardson*² 1) Faculty of Applied Medical Sciences, University of Hafr Al-Batin, Kingdom of Saudi Arabia; 2) Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, University of Manchester, UK.

**Objective:** Fungi are common members of the microbiota of the indoor environment and constitute a serious public health threat in homes and workplaces. A major component of the fungal mycobiome in these environments is *Aspergillus fumigatus* which is responsible for a spectrum of pulmonary diseases

The present study was designed to investigate the diversity of allergenic and pathogenic fungi in the settled dust of homes of patients with chronic pulmonary aspergillosis to assess levels of exposure and disease severity. Dust is a valuable matrix to analyse since it reflects the history of the patients' home.

**Methods:** Dust samples were collected from the vacuum cleaners of control and patients' houses and analysed by conventional culture and molecular techniques. Sequencing of ribosomal DNA from environmental fungi offers a valuable tool for analyzing the mycobiome of indoor environments. DNA extracted from 21 dust samples as amplified and analysed using the ITS-1 forward primer and the ITS-4 reverse primer.

**Results:** Based on conventional culture methods a high diversity of environmental fungi was detected. It was possible to identify three different species of Aspergillus: *A. niger*, *A. fumigatus* and *A. flavus*. *Rhizomucor*, *Penicillium* and *Mucor*, were also detected. In addition to filamentous fungi, a variety of yeasts were present. *A. fumigatus* was present in the majority of houses. The ITS region of rDNA was amplified by PCR which confirmed the presence of fungi in our dust samples.

**Conclusions:** The analysis of settled dust by PCR provides a sensitive technique for the investigation of the diversity of the fungal mycobiome in indoor environments and will potentially identify houses where fungal exposure by patients with aspergillosis needs to be
controlled. Morphological characterizations and molecular techniques such as PCR were used to further confirm the identity of the
mycobiome of dust. Current work includes microbiome analysis by next - gen sequencing.

**319W Field dynamics and patho-genomics of Asian soybean rust pathogen *Phakopsora pachyrhizi*.** Y.K. Gupta¹, J. Ku¹, D MacLean¹, H.M. Murithi³,⁵, M.H.A.J. Joosten⁵, G. Tabor⁴, K.E. Broglie⁴, S.H. Brommonnschenkel², H.P van Esse¹ 1) The Sainsbury Laboratory, Norwich, Norfolk, GB; 2) Dep. de Fitopatologia, Universidade Federal de Viçosa, Viçosa, Brazil; 3) International Institute of Tropical Agriculture (IITA), Dar es Salaam, Tanzania; 4) Agricultural Biotechnology, DuPont-Pioneer, Wilmington, Delaware, USA; 5) Laboratory of Phytopathology, Wageningen University, Wageningen, Netherlands.

Soybean (*Glycine max*) is an important legume crop and a rich source for plant proteins and vegetable oil. A major constraint for soybean production is Asian soybean rust (ASR) which causes yield losses of up to 80%. ASR is caused by the obligate biotrophic fungus *Phakopsora pachyrhizi* and can only be controlled by fungicide application. Extensive efforts have been made to find genetic resistance against *P. pachyrhizi* however, the pathogen has rapidly overcome major resistance genes *Rpp1-5*. Remarkably, *P. pachyrhizi* infects over 153 different legume species under suitable conditions, which suggests that *P. pachyrhizi* maintains diverse pathogenicity factors. The degree and distribution of genetic variation in the pathogen population is key for informed deployment of novel resistance genes but is largely unknown for *P. pachyrhizi*. To understand the field variation and population structure of *P. pachyrhizi*, infected field samples of soybean are collected from different locations across Brazil and east Africa. We have developed a next-generation sequencing approach to explore the population structure and genetic variation of *P. pachyrhizi* in these very different geographic locations. Sequencing of field samples will allow us to study sequence polymorphisms in *P. pachyrhizi*. We will aim to obtain a detailed understanding of the effector diversity in the field population of *P. pachyrhizi*. We will use this information for the sustainable deployment of genes that confer resistance to Asian Soybean Rust in transgenic soybean.

**320T Genomic and phenotypic population heterogeneity of *Candida lusitaniae* in chronic lung infections.** *E. Demers*¹, S. Masonjones², N. Grahl¹, A.R. Biermann¹, A.W. Crocker¹, J.E. Stajich², D.A. Hogan¹ 1) Geisel School of Medicine, Dartmouth College, Hanover, NH; 2) University of California, Riverside, CA.

Despite the fact that fungal isolates recovered from infections frequently test as sensitive to antifungal drugs *in vitro*, chronic fungal infections are notoriously hard to treat. The mechanisms behind this resistance are not well understood, though recent studies of bacterial infections and human tumors are starting to acknowledge that heterogeneity within these populations may lead to an alteration in our assessment of drug susceptibility from testing one isolate. We have collected numerous isolates from three subjects with cystic fibrosis who have chronic, high fungal burden infections; we propose these represent an important model for studying population heterogeneity in chronic fungal infections. We identified the fungus in all three subjects (A, B and C) as *Candida lusitaniae*, which is an uncommon pathogen but has been previously studied because of its ability to rapidly develop resistance to amphotericin B and more recently echinocandins. Using a mixture of Illumina sequencing technology and phenotypic assays we have characterized numerous isolates from three subjects for genome sequence heterogeneity, MIC for a variety of drugs, growth rate and metabolic activity. Interestingly, we observed a high level of heterogeneity in drug resistance in all three subjects for multiple classes of antifungals, despite the fact that none of the subjects has received antifungal therapies in the previous year. Additionally, whole genome sequencing analysis revealed a high level of heterogeneity within each subject's fungal population, with at least 100-1000 SNPs varying among isolates from an individual subject when compared to a reference genome. Comparison of phenotypic and genomic analysis among Subject A isolates revealed a correlation between one of the most variable loci, *MRR1*, which encodes a known regulator of antifungal resistance, and fluconazole (FLC) MIC, which in some isolates was quite high. In addition, both deletion of *MRR1* from the clinical isolate D2Y8 and analysis of mating progeny from mating with a highly FLC sensitive strain, indicated that *MRR1*<sup>D2Y8</sup> confers high FLC resistance. These studies may prompt a reexamination in the way in which we view and study chronic fungal infections, introducing the need to examine multiple isolates from individuals to accurately assess infecting populations and potentially altering treatment strategies to account for isolates with heterogeneous drug resistance harbored within the population.

**321F Patterns of mycofloral diversity and composition in Korean traditional fermentation starter *nuruk* as influenced by fermentation temperature and external humidity.** Jyotiranjan Bal¹, Suk-Hyun Yun¹, Soo-Hwan Yeo², Jung-Mi Kim³, *Dae-Hyuk Kim*¹ 1) Department of Molecular Biology, Institute for Molecular Biology and Genetics, Chonbuk National University, Jeonju, Republic of Korea; 2) Fermented Food Science Division, Department of Agrofood Resource, NAAS, RDA, Wanju-gun, Republic of Korea; 3) Department of Bio-Environmental Chemistry, Institute of Life Science and Natural Resources, Wonkwang University, Iksan, Republic of Korea.

The traditional Korean fermentation starter *nuruk* is typically composed of moistened coarsely ground cereals, packed into a mold, and left to ferment. Korea's popular alcoholic beverages are brewed using *nuruk*. Emerging popularity of Korean alcoholic beverages demands for its quality enhancement. The *nuruk* mycobiome greatly influences both fermentation and palatability enhancement. In our previous experiments, we have assessed the mycofloral diversity across varying temperatures and internal moisture contents of *nuruk*. In continuation of our efforts to explore traditional *nuruk* mycobiome, we further evaluated mycofloral dynamics of two barley based-*nuruk* samples (E and F) consisting of barley and green gram, fermented under 30°C and 40°C, respectively, for a period of 30 days. The initial external humidity was maintained at 70% for three days following which it was reduced to 50%. The average observed fungal richness during the 30 day *nuruk* fermentation was higher in *nuruk* E, as compared to *nuruk* F. Variations in both abundance and diversity of mycoflora was observed with different fermentation temperatures as well as changes in external humidity. Day 6 of *nuruk* fermentation revealed greater abundance of *Saccharomyces* *fibuligera*. The genera *Saccharomyces* predominantly inhabited both *nuruks*. However, *Aspergillus* dominated *nuruk* E and *Rhizopus* dominated *nuruk* F. In *nuruk* E at 70% humidity, *Aspergillus* predominated whereas *Saccharomyces* at 50%. With increase in fermentation duration, *Aspergillus* abundance increased whereas that of *Saccharomyces* decreased. Most of the mycofloral communities present abundantly in the 0 day samples either perished or diminished with the progress in fermentation. These findings are a step forward in our aim of understanding *nuruk* microbiome and thereby its quality enhancement.
**322W Fungal diversity of marine sediments from the Gulf of Mexico.** L. Vargas Gastelum¹, A.L. Romero-Olivares⁶, J. Catalán-Dibene⁷, A. Rocha-Olivares², C. Brizuela³, S. Herzka⁴, A.E. Escalante⁵, M. Riquelme¹
1) Department of Microbiology, Centro de Investigación Científica y de Educación Superior de Ensenada, CICESE, Mexico; 2) Department of Marine Ecology, CICESE, Ensenada, Mexico; 3) Department of Computer Sciences, CICESE, Ensenada, Mexico; 4) Department of Biological Oceanography, CICESE, Ensenada, Mexico; 5) Instituto de Ecología, Universidad Nacional Autónoma de México UNAM, Ciudad Universitaria, Mexico; 6) Departments of Ecology and Evolutionary Biology, University of California-Irvine, California, USA; 7) Physiology and Biophysics, University of California-Irvine, California, USA.

Fungi inhabiting marine sediments interact with other organisms and contribute to the biogeochemical cycling of elements. These important ecological processes are susceptible to anthropogenic disturbances such as oil spills. The impact of these disturbances is only measurable if the habitat was characterized prior to the disaster. Our aim was to establish the baseline of the benthic mycobiota of the Mexican Exclusive Economic Zone of the Gulf of Mexico in order to detect changes of these communities as a result of future potential disturbances. To achieve this, ten stations between parallels 19 to 25°N and 86 to 97°W were sampled. Total genomic DNA was recovered and used as template to amplify the ITS region by PCR followed by Illumina MiSeq sequencing. A total of 2,051,282 sequences were recovered and analyzed, resulting in 1,149 OTUs. The fungal community appears to be influenced by the geographic location (Zone 3, deepest stations; Zone 4, stations closer to the coast). Zones 3 and 4 shared only 68 OTUs. Zones 3 and 4 displayed 72 and 55 unique OTUs, respectively, indicative of specific functions correlated to the underlying characteristics of each zone. The remaining 954 OTUs were shared between two or more stations. The Ascomycota and the Basidiomycota represented the main components of the community. The most abundant orders were Sordariales and Malasseziales. A group of unidentified sequences, *Neurospora terricola*, *Malasseziales* sp. and *Capnobotryella* sp. were the most abundant phylotypes. A wide variety of the identified fungi have been described as facultative marine fungi (derived from terrestrial ecosystems). Ongoing annual samplings will allow us to expand our understanding of the deep-sea fungal community.

**323T Diversity of Spalting Fungi in the Peruvian Amazon.** Sarath Vega Gutierrez, Sara Robison Wood Science and Engineering, Oregon State University, Corvallis, OR.

Most of the research related to fungi of the Peruvian Amazon is focused on edible mushrooms and pathogens. Other important fungi, such as spalting fungi, are not broadly studied because most of them do not produce fruiting bodies, are difficult to locate, and do not present an immediate value to the forager. Finding spalting fungi involves a complete sampling of downed trees and branches in a given area through the opening of the xylem to identify potential pigmenting and zone line producing fungi. This methodoly was employed in the district of Las Piedras, Madre de Dios, Peru. Fungi suspected of causing internal pigment and zone lines were collected, cultured, isolated, and sequenced. The species found belonged to the orders of the Helotiales, Xylariales, Hypocreales, Russulales, Polyporales, Botryosphaeriales and two specimens of the class Leotiomycetes. Most of the fungi produced pigments or zone lines in wild conditions and all of them were part of the wood decomposing fungi. Findings from this research will benefit Amazonian communities as well as Peru as whole, through identification and location of value-added wood products that can widen the commercial wood market currently focused on dark, old growth trees. This also gives more inherent value to the forest, potentially saving the land from being converted to plantation or pasture.

**324F Hidden diversity in the oomycete genus Olpidiopsis is a global threat to red algal cultivation.** Y. Badis¹, M. Strittmatter¹, P. Murua¹, g.h Kim², c Gachon¹
1) Microbial and Molecular Biology, Scottish Ass. for Marine Science, Oban, Argyllce; 2) Dept of Biology, Kongju National University, Kongju, Korea.

Seaweed cultivation is the **fastest-growing of all aquaculture sectors**, with an annual growth rate of 8 % and a value in excess of $5bn. Emerging diseases are a threat to sustainable development. The oomycete genus *Olpidiopsis* notably harbours pathogens of red algae, and include the most economically damaging disease of *Pyropia* (ex-*Porphyra*) farms in Asia. Using microscopy and molecular analysis, we identified in Scotland three new *Olpidiopsis* species: *Olpidiopsis palmariae*, *O. muelleri*, and *O. polysiphoniae* spp. nov. A Scottish variety of *Olpidiopsis porphyrae*, a devastating pathogen previously thought to be restricted to Japanese seaweed farms, is also described as *O. porphyrae* var. *scotiae*. Importantly, two of the new species infected the red seaweed *Porphyra* and *Palmaria* sp, which are subject extensive farming trials in Europe and North America. To further assess the extent of undescribed *Olpidiopsis* diversity and the threat it might pose to aquaculture in different regions, a metagenomics survey of eukaryotes associated from *Porphyra umbilicalis* thalli collected across the East and West Northern Atlantic shores was conducted, and combined to publicly available environmental sequences. Screening efforts were extended to global metagenomics barcoding campaigns Over 700 new *Olpidiopsis* sequences were detected with a truly worldwide distribution and grouped in three major Olpidiopsis clades. Close relatives of the Korean *O. pyropiae* are also reported for the first time in Europe and the United States. In the light of our restricted sampling, our results highlight the diversity and abundance of *Olpidiopsis* associated to red macroalgae worldwide, most of which are undescribed. In the context of worsening impact of *Olpidiopsis* pathogens in Asia, where red algal cultivation has grown steadily over the last two decades, this worldwide distribution should be treated as a serious threat to the global seaweed industry, and to the conservation of wild red algal populations near farms. Our data calls for more efforts towards the documentation of these pathogens, and for adequate biosecurity measures to be developed.

**325W Microbial communities associated with water-damaged New York City public housing residences.** Iman Sylvain¹, John Taylor¹, Michal Spilak², Michael Waring², Rachel Adams¹
1) Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA, USA; 2) Department of Civil, Architectural and Environmental Engineering, Drexel University, USA.

The use of high-throughput sequencing technologies has elucidated the high biodiversity of microorganism in the built environment. Fungi found indoors in healthy buildings appear to be a random subset of the diversity found outdoors, but our knowledge of fungi associated with unhealthy buildings indoors is still quite limited, and largely informed by culture-based studies. To date a number of studies have shown that as water availability indoors increases, fungal biomass and richness indoors also increases. Damp, moldy housing is associated with a number of adverse health outcomes, principally asthma and respiratory symptoms, as well as anxiety and depression. It is well-established that housing quality is a major social determinant of health, and differential access to adequate housing is believed to be the fundamental cause of ethnic/racial health disparity in America.
Since Hurricane Sandy hit the Eastern Seaboard in 2012, the presence of mold has been a major problem in New York City public housing. In this study we have used high-throughput sequencing technologies to compare the indoor microbioata of New York City public and private residences impacted by Hurricane Sandy. Using quantitative-PCR and Illuminia MiSeq we have assessed community composition, diversity, and biomass of fungi and bacteria found in the house dust of 60 residential units in Brooklyn and Manhattan, over two seasons. Our microbiological analysis was paired with records of dwelling characteristics, environmental parameters, and occupant behavior over the course of one-month.

By amplifying the ITS1 and 16S rRNA regions for fungi and bacteria in over 600 dust samples collected from vacuums, swabs, electrostatic wipes, and empty petri dishes, we see that there are distinct microbial communities indoors in public and private apartments in New York City. Results showing significant differences in fungal community composition in public and private housing were also observable at the neighborhood level, i.e. within Brooklyn and Manhattan. While public housing had greater observable water-damaged than private housing, the presence or absence of water-damage did not appear to be the major factor driving differences in microbial communities across units or boroughs. The long-standing distinction in building maintenance in public and private housing seems to pre-date the water-damage caused by the hurricane, and has impacted both the microbial and human occupants.

**326T Evolution of host preference and sorus location in the plant parasitic genus *Microbotryum*.** Martin Kemler¹, Dominik Begerow¹, Matthias Lutz²
1) Ruhr-Universität Bochum, Bochum, DE; 2) Eberhard Karls Universität Tübingen, Tübingen, DE.

*Microbotryum* is a basidiomyceteous genus known for being the causing agent of anther smut in Caryophyllaceae. Monophyletic genera within the Caryophyllaceae recurrently have been colonized once, with subsequent radiation and specialization to many different host species. The host range of *Microbotryum* however is considerably larger than Caryophyllaceae and includes host species in many other plant genera and families. Additionally, other locations of sorus formation than anthers have evolved. In this study we present an updated phylogenetic approach using a denser sampling of species from different host families. The questions of this study are: 1) What is the ancestral state of host genus parasitized by the genus *Microbotryum*? 2) What is the ancestral state of sorus location in the genus *Microbotryum*? 3) Are monophyletic clades of parasites found on monophyletic groups of hosts? Our results indicate and corroborate previous results that the ancestor of the genus evolved on Polygonaceae and that most likely it already formed its spores in inflorescences. As in the Caryophyllaceae, many monophyletic groups of hosts have been colonized once with subsequent radiation. These results will be discussed in the light of some life-history traits of hosts and parasites.

**327F Biodiversity and phylogeny of *Marasmius* (Agaricales, Basidiomycota) from Madagascar.** J.E. Shay¹, D.E. Desjardin¹, B.A. Perry², C.L. Grace¹, D.S. Newman¹
1) Department of Biology, San Francisco State University, 1600 Holloway Ave., San Francisco, CA 94132, USA; 2) Department of Biological Sciences, California State University East Bay, 25800 Carlos Bee Blvd., Hayward, CA 94542 USA.

Prior to this monographic treatment, limited research on the genus *Marasmius* (Basidiomycota, Agaricales) had been conducted in Madagascar. Based on field work in January 2013 and January–February 2014, which generated 45 specimens of *Marasmius sensu stricto*, supplemented by herbarium exsiccatae and published literature, 35 species of *Marasmius* were documented from Madagascar. Of these, a few key species are selected to demonstrate the variation in micro- and macromorphology of *Marasmius* from Madagascar. Line drawings of salient micromorphological features and color photographs of basidiomes are provided for selected species. Species delineations are based on comprehensive morphological descriptions and molecular sequence data from the internal transcribed spacer (ITS) region.

**328W Diversity and Genome Analysis of Marine Fungi.** F. Kempken, P. Phule, A. Kumar Botanisches Institut, Christian-Albrechts University, Kiel, DE.

The number of species and distribution of marine fungi is largely unknown as marine fungi have been much neglected for a long time, although respective studies were published decades ago (1). Current estimate for numbers of marine fungal species is over 10,000 (2), but may be much higher (3). Nevertheless, their role in marine ecology is elusive (4).
To investigate the number of cultivatable fungi from marine sediments we have analyzed 16 samples from the Baltic Sea seafloor with a depth of 150 to 350 m. We were able to obtain 60 fungal species including isolates of *Chaetomium*, *Emericellopsis*, *Microascus*, *Penicillium*, *Phoma*, *Preussia*. The genome of the Preussia isolate has recently been sequenced and is currently being annotated. In sediment from the Mid-Atlantic ridge we did identify yeast and filamentous fungi. Several other fungi were isolated from beaches of the Baltic Sea in Germany, and the US west coast. Among the latter, *Asteromyces cruciatus* was re-identified, which previously had been described as a marine fungus.

At current, we have sequenced and annotated four marine genomes, *Scopulariopsis brevicaulis* (5) (from a mediterreanean sponge), *Pestalotiopsis* sp., *Calcarisporium* sp., and a new *Fusarium* species from the Baltic Sea (unpublished). The enormous biodiversity of marine fungal isolates is mirrored by the molecular diversity of their secondary metabolites (6–8). Yet, these studies are mainly chemistry based, and marine fungi remain tremendously underexplored with regard to species, distribution and applications (9, 10). We report here 44 NRPSs, 62 PKSs and seven hybrid NRPS-PKS genes for *Pestalotiopsis* sp. and 52 NRPSs, 66 PKSs and seven hybrid NRPS-PKS genes for *Calcariosporium*. This number of secondary metabolites is enormous and emphasis the potential of marine fungi for the discovery of new metabolites.

1. Kohlmeyer J, Kohlmeyer E. 1979. Academic, New York.

2. Jones EBG. 2011. Bot Mar 54:343–354.

3. Mora C, T et al. .. 2011. H PLoS Biol 9:e1001127.

4. Richards TA, et al. 2012. Ann Rev Mar Sci 4:495–522.

5. Kumar A, ..., Kempken F. 2015. PLoS One 10: 10.1371/journal.pone.0140398

6. Bugni TS, Ireland CM. 2004. Nat Prod Rep 21:143–63.

7. Saleem M, et al. 2007. Nat Prod Rep 24:1142–1152.
8. Konig GM, et al. 2006. Chembiochem 7:229-238.

9. Rateb ME, Ebel R. 2011. Nat Prod Rep 28:290-344.

10. Debbab A, et al. 2010. Bioactive Microb Biotechnol 3:544-563.

**329T Evolution and diversity of *Monosporascus* in arid-land environments.** A.J. Robinson, D.O. Natvig, M.I.

Hutchinson Department of Biology, University of New Mexico, Albuquerque, NM.

The genus *Monosporascus* represents an enigmatic group of fungi important in agriculture and widely distributed in arid ecosystems. Only three species of *Monosporascus* are currently described, and two are devastating pathogens on the roots of cantaloupe and other cucurbits. Nearly all research on this genus is limited to agricultural settings although members of the genus are nearly ubiquitous in surveys of root endophytes in arid environments of the southwestern United States and Europe. We have focused on isolates obtained from the roots of grasses, shrubs and herbaceous plants from central New Mexico. Comparison of the ribosomal internal transcribed spacer (ITS) DNA region and other genes indicates substantial diversity among strains. The New Mexico isolates include close relatives of the three described species and strains that appear to represent previously unrecognized lineages. There currently is no evidence for pathogenicity among any of the New Mexico lineages. To resolve the evolutionary relationships within the genus and the placement of the genus within the Ascomycota, we have sequenced and assembled the genomes of *M. cannonballus*, *M. ibericus* and several isolates obtained in New Mexico. Laboratory experiments suggest important life-cycle differences between described species and isolates from New Mexico. In contrast with *M. cannonballus* (homothallic), sexual reproduction has not been observed among any of the New Mexico isolates. Preliminary characterization of the genomes of 10 strains indicates that the genus likely belongs to the Xylariales, not to the Sordariales where it has generally been placed. The genomes of the isolates are consistently substantially larger than what is typical of other members of the Sordariomycetes and also raise interesting questions about life cycle biology and sexual reproduction. To date, we have failed to identify in these genomes clear homologs of any of the three genes associated with mating type A (Mat 1-1) in other heterothallic and homothallic filamentous Ascomycota.

**330F Study on diversity of root-associated fungi of an Orchid *Crepidium acuminatum* (D. Don) Szlach: A rare and medicinally important taxon.** J. Thakur, P. Uniyal Department of Botany, University of Delhi, India, New Delhi, Delhi, IN.

Mycorrhizal association is known to be essential for seed germination and seedling establishment in majority of orchids. Orchids possess embryo which lacks endosperm, the food reservoir. Hence, orchid roots harbour a large diversity of fungal species to procure carbon and other mineral nutrients to complete their life cycle. *Crepidium acuminatum* (Orchidaceae) is a rare medicinal plant that grows under shady and moist forest floor where light remains for a very short period of time. Thus, it is speculated that light is one of the key factors that enforces this species to form symbiotic association with diverse group of fungi. Identification of fungi that form mycorrhizae with orchids is of crucial importance for orchid conservation. We used both morphological as well as molecular approaches to study this plant-fungal interaction. Scanning electron microscopy showed that fungi grow and proliferate in the middle layers of cortex. Also, spiral root hairs were found along with roots hairs, which is an unusual observation. We believe that spiral root hairs improve fluid absorption, entrance of colonisers and increase adherence to the substrate. Furthermore, root samples were collected from four states in India. Fungal genomic DNA was isolated and fungal ITS regions were PCR-amplified using primer combinations ITS1F/ITS4 and ITS1/ITS4tul. ITS sequences were analyzed with BLAST against the NCBI sequence database to know the closest sequence matches in the GenBank database. Samples were identified to be belonging to three main genera i.e., *Tulasnella*, *Aspergillus* and *Penicillium*. Results indicate that mycorrhizal association is necessary for the growth and development of plant. In addition, these symbiosis influence the distribution and rarity of this medicinally valuable taxon. Specific fungal partners may lead to enhanced seed germination rate and increased efficiency of nutrient exchange between both the partners. Hence, knowledge of mycorrhizal fungi is essential for future *in vitro* germination and seeding establishment programs, because they rely on fungi for germination. Identification of mycorrhizal fungi can be used for orchid propagation, conservation and reintroduction in natural habitats.

**331W Dissecting intraspecies diversity in fungal wood decay.** S. Casado Lopez¹, S Purvine², A Dohnalkova², G Orr², E Robinson², E Zink², S Baker², K Hildén³, M Mäkelä³, R de Vries¹ 1) Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre, Utrecht, NL; 2) Environmental Molecular Sciences Laboratory, Department of Energy, USA; 3) Department of Food and Environmental Sciences, University of Helsinki, Finland.

*Dichomitus squalens* is a white rot fungus that colonizes mainly softwood and is commonly found in the northern parts of Europe, North America and Asia. In nature, compatible monokaryons of *D. squalens* cross sexually to form a dikaryon. At this point it is not clear whether monokaryons can efficiently colonize and degrade wood, or whether this only occurs after the dikaryon is formed.
Here we studied in detail the process of wood colonization, degradation and utilization by *D. squalens*, using a combination of transcriptomics, proteomics, metabolomics and high-resolution microscopy enabled by the combined facilities of the JGI and EMSL institutes.

In this study, we compared a set of related mono- and dikaryotic strains, originating from two isolates. All strains were grown on solid spruce sticks and sampling was performed after two and four weeks to evaluate the variation between the strains during the wood decay process. Monokaryons, compared to dikaryons, were notably slower in colonizing wood, which was reflected in the analysis of the various datasets obtained from the samples.

**332T Heterokaryon incompatibility and phenotypic characterisation of *Aspergillus flavus* isolates in low and high risk zones in Kenya.** Alfred Mitema¹,², Sheila Okoth², Revel Lyer², Amelia Hilgart², Suhail Rafudeen² 1) Molecular and Cell Biology, University of Cape Town, Cape Town, Western Cape, ZA; 2) University of Nairobi, Kenya.

Species *Aspergillus flavus* (*A. flavus*) has three morphotypes: S-, L- and S<sub>BG</sub>-type strains, with the first repeatedly being associated with acute aflatoxicosis. Aflatoxicosis in Kenya is a serious health problem with outbreak 317 cases and 215 deaths. In 2013, we choose to address this crisis by characterising the diversity of *A. flavus* isolates through comparison of vegetative compatibility groups (VCGs), phenotypic characteristics and mycotoxin profiles across various agricultural regions in Kenya where aflatoxicosis has occurred previously. Using diagonal transect random household sampling, Maize kernels were collected from Makueni, Homa Bay, Nandi, and Kisumu counties
between November and December 2013. Out of 37 isolates, the nitrate non-utilising auxotrophs (nit mutants) complementation test revealed 20 VCGs. KVCG14 and KVCG15 had highest distribution frequency (n = 13; 10.8%) while KVCG10 and KVCG20 (n = 1; 0.8%) the least. Analyses of VCG diversity using the Shannon Index (H) showed that Nandi (H' = 0.108) and Kisumu (H' = 0.324) counties recorded the lowest and highest VCG diversities respectively. Results also showed that, with a few exceptions, within-county isolates were self-compatible but they were incompatible across any two adjacent counties and across all the sampled counties. Heterokaryon incompatibility in Nandi (n = 6; 67%) and Makueni (n = 3; 33%) were the greatest and least respectively. Mycotoxin detection by coconut cream agar under UV light (365 nm) revealed blue fluorescence ((57%, n = 21) aflatoxin B) and green ((43%, n = 16) aflatoxin G). The study further revealed L-, S- type strains (57%; n = 21 and 7%; n = 2) respectively. The findings of the study could provide reliable information in determining biocontrol strategies to mitigate aflatoxin contamination of maize by A. flavus in the studied counties.

**333F Mycobiome in Anorexia Nervosa.** K. Mueller¹, E. Huh¹, E. Bulik-Sullivan³, H. Zhang¹, Y. Wang¹, C. Bulik³,⁴,⁵, I. Carroll³, S. Lee¹,² 1) Biology, The University of Texas at San Antonio, San Antonio, TX; 2) South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, TX; 3) Medicine, The University of North Carolina at Chapel Hill, NC; 4) Nutrition, The University of North Carolina at Chapel Hill, NC; 5) Psychiatry, The University of North Carolina at Chapel Hill, NC.

Anorexia nervosa(AN) is a mental illness characterized by a fear of gaining body weight, which results in severe weight loss and malnutrition. Persistent malnutrition, along with anxiety as a common comorbidity, contributes to AN having the highest mortality rate of mental illnesses. Typical treatment for AN involves re-nourishment of patients, which has proven to be insufficient in treating the disease, as relapse rates after treatment reach 50%. As such, finding new treatments for AN is of great importance.

Previous studies have shown an overall lowered bacterial diversity in AN patients, as well as a significant association between the bacterial diversity of AN patients and anxiety. However, these studies have overlooked the possible role of fungal diversity in the gut. Intestinal fungi have been implicated in diseases of the gastrointestinal tract, and previous studies have shown that disruption of normal gut bacteria may lead to enteric fungal dysbiosis. It then stands to reason that the fungal gut community, as well as an interplay between the fungal and bacterial communities, also plays a role in AN associated dysbiosis.

DNAs extracted from stool samples were used to analyze mycobiota in the guts of AN patients and healthy controls (HC). Utilizing fungal specific ITS1 primers and bacterial specific 16s rDNA primers, qPCR analysis demonstrated a significant increase in number of gut fungi relative to bacteria in the undernourished phase (T1) of AN patients as compared to the post-refeeding phase (T2) of the same patients. Furthermore, an increase in *C. albicans* was observed upon patients undergoing the refeeding treatment, providing a specific role of this fungus in lower weight phase of AN patients. Illumina sequencing of the ITS1 amplicons was performed to examine the dynamics of fungal species present in AN patients and HC. The obtained sequence data were filtered to identify taxa via IPITS pipeline. The OTU abundance from each sample was assessed with QIIME. Measurements of alpha diversity (T1 vs T2 vs HC) with the number of species and the Chao-1 index revealed less fungal species present in the gut of AN patients compared to that of HC. This result is similar to previous studies considering lower bacterial diversity in AN. Together, these findings suggest that fungal dysbiosis in the intestines may play a part in perpetuating microbiota in AN patients. Addressing this dysbiosis may then offer new methods for the treatment of AN.

**334W Nothophytophthora prov. nom., a new sister genus of Phytophthora.** T. Jung¹,²,³, B. Scanu⁴, J. Bakonyi⁵, D. Seress⁵, A. Durán⁶, E. Sanfuentes von Stowasser⁷, L. Schena⁸, S. Mosca⁸, P. Q. Thu⁹, C. N. Minh⁹, S. Fajardo⁷, M. González⁷, A. Pérez-Sierra¹⁰, H. Rees¹⁰, C. Maia¹, *M. Horta Jung*¹,² 1) Laboratory of Molecular Biotechnology and Phytopathology, Center for Mediterranean Bioresources and Food (MeditBio), University of Algarve, 8005-130 Faro, Portugal; 2) Phytophthora Research Centre, Mendel University, 613 00 Brno, Czech Republic; 3) Phytophthora Research and Consultancy, 83131 Nußdorf, Germany; 4) Dipartimento di Agraria, Sezione di Patologia vegetale ed Entomologia (SPaVE), Università degli Studi di Sassari, 07100 Sassari, Italy; 5) Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, 1022 Budapest, Hungary; 6) Ontario Forest Research Institute, ?P6A 2E5 Sault Ste. Marie, Canada; 7) Laboratorio de Patología Forestal, Facultad Ciencias Forestales y Centro de Biotecnología, Universidad de Concepción, Concepción, Chile; 8) Dipartimento di Gestione dei Sistemi Agrari e Forestali, Università Mediterranea di Reggio Calabria, 89124 Reggio Calabria, Italy; 9) Forest Protection Research Centre, Vietnamese Academy of Forest Sciences, Duc Thang Ward, Northern Tu Liem District, Hanoi, Vietnam; 10) Forest Research, Alice Holt Lodge, Farnham, Surrey GU10 4LH, United Kingdom.

During various surveys of *Phytophthora* diversity in Europe, Chile and Vietnam slow growing oomycete isolates were obtained from soil samples and small streams in natural and planted forest stands. Phylogenetic analyses of ITS, β-tubulin, LSU and *cox1* sequences placed them into six new species belonging to a new genus, officially described as *Nothophytophthora* gen. nov., which clustered sister to *Phytophthora*. *Nothophytophthora* species share numerous morphological characters with *Phytophthora*: persistent (all *Nothophytophthora* spp.) and caducous (*N. caduca*, *N. chlamydospora*, *N. valdiviana*, *N. vietnamensis*) sporangia with variable shapes, internal differentiation of zoospores and internal, nested and extended (*N. chlamydospora*, *N. caduca*) and external (all *Nothophytophthora* spp.) sporangial proliferation; smooth-walled oogonia with amphigynous (*N. amphigynosa*) and paragynous (*N. amphigynosa*, *N. intricata*, *N. vietnamensis*) insertion of the antheridia; chlamydospores (*N. chlamydospora*) and hyphal swellings. Comparisons of morphological structures of both genera allow clues about the morphology and ecology of their common ancestor which are discussed. Including *Nothophytophthora* in coalescence analyses will give new insights into the evolutionary history of *Phytophthora*. Production of caducous sporangia by *N. caduca*, *N. valdiviana* and *N. chlamydospora* from Valdivian rainforests and *N. vietnamensis* from a wet mountain forest in Vietnam indicates a partially aerial lifestyle as adaptation to these humid habitats. Presence of tree dieback in all forests from which *Nothophytophthora* spp. were recovered and partial sporangial caducity of several *Nothophytophthora* species suggests they may be facultative pathogens. Pathogenicity tests are urgently required to clarify whether the individual *Nothophytophthora* species have a pathogenic or saprophytic lifestyle.

**335T Diversity of Phytophthora species from natural and semi-natural ecosystems in Portugal, Chile and Vietnam.** T. Jung¹,²,³, B. Scanu⁴, J. Bakonyi⁵, D. Seress⁵, A. Durán⁶, E. Sanfuentes von Stowasser⁷, L. Schena⁸, S. Mosca⁸, P. Q. Thu⁹, C. N. Minh⁹, S. Fajardo⁷, M. González⁷, A. Pérez-Sierra¹⁰, H. Rees¹⁰, C. Maia¹, B. Mora Sala¹¹, G. Carella¹², S. Moricca¹², A. Cravador¹, *M. Horta Jung*¹,² 1) Laboratory of Molecular Biotechnology and Phytopathology, Center for Mediterranean Bioresources and Food (MeditBio), University of Algarve, 8005-130 Faro, Portugal; 2) Phytophthora Research Centre, Mendel University, 613 00 Brno, Czech Republic; 3) Phytophthora Research and Consultancy, 83131 Nußdorf; 4) Dipartimento di Agraria, Sezione di Patologia vegetale ed Entomologia (SPaVE), Università
degli Studi di Sassari, 07100 Sassari, Italy; 5) Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, 1022 Budapest, Hungary; 6) Ontario Forest Research Institute, ?P6A 2E5 Sault Ste. Marie, Canada; 7) Laboratorio de Patología Forestal, Facultad Ciencias Forestales y Centro de Biotecnología, Universidad de Concepción, Concepción, Chile; 8) Dipartimento di Gestione dei Sistemi Agrari e Forestali, Università Mediterranea di Reggio Calabria, 89124 Reggio Calabria, Italy; 9) Forest Protection Research Centre, Vietnamese Academy of Forest Sciences, Duc Thang Ward, Northern Tu Liem District, Hanoi, Vietnam; 10) Forest Research, Alice Holt Lodge, Farnham, Surrey GU10 4LH, United Kingdom; 11) Instituto Agroforestal Mediterráneo, Universitat Politècnica de Valencia, Edificio 3H, Camino de Vera s/n, 46022 Valencia, Spain; 12) Department of Agri-Food Production and Environmental Sciences, Plant Pathology and Entomology Division, University of Florence, Piazzale delle Cascine, 28, 50144 Florence, Italy.

Surveys of *Phytophthora* diversity were performed using baiting assays and direct plating of necrotic plant tissues. Isolates were identified using both classical identification and sequence analysis of ITS and *cox1*.

In Portugal, 68 forest stands, 12 forest nurseries, 38 river systems and 4 lagoon ecosystems were surveyed. The isolates obtained belonged to 29 known species, 3 informally designated taxa, and1 previously unknown taxon of *Phytophthora*, nine new *Phytophthora* hybrid taxa from Clades 6 and 9, *Nothophytophthora homothallica* nom. prov., 1 known and 10 new *Halophytophthora* species, 7 known species and one new taxon of *Phytopythium*, and multiple *Pythium* species were isolated.

In Chile, the survey was performed in 13 natural forest stands and 20 forest streams located in two protected areas near Valdivia and in a temperate mountain forest in the Concepción area, and in each one planted stand of the introduced tree species *Castanea sativa* and *Fagus sylvatica*. Eight described species (including *P. kernoviae*) and 2 previously unknown taxa of *Phytophthora* were isolated. In addition, a diverse array of Clade 6 hybrids, and *Nothophytophthora caduca* nom. prov., *Nothophytophthora chlamydospora* nom. prov. and *Nothophytophthora valdiviana* nom. prov., were obtained.

In Vietnam the survey was performed in 23 natural forest stands and 10 forest streams and rivers in temperate montane and tropical lowland regions, and in 14 rubber plantations. Sixteen described species (including *P. ramorum*), 3 designated taxa and 23 previously unknown taxa of *Phytophthora*, amongst them 9 Clade 9 hybrid taxa, were isolated. In addition, *Nothophytophthora vietnamensis* nom. prov. and a diverse array of known and new taxa of *Phytopythium*, *Pythium* and *Elongisporangium* were recovered.

The implications of these findings for plant biosecurity and the development of a deeper understanding of the evolution and adaptability of the genus *Phytophthora* will be discussed

**336F Fungi challenge global food security.** Sarah Gurr, Dan Bebber, Helen Fones Biosciences, Exeter University, Exeter, GB.

Over the past centuries, crop diseases have led to the starvation of the people, the ruination of economies and the downfall of governments. Of the various challenges, the threat to plants of fungal infection outstrips that posed by bacterial and viral diseases combined. Indeed, fungal diseases have been increasing in severity and scale since the mid. 20th Century and now pose a serious threat to global food security and ecosystem health.

We face a future blighted by known adversaries, by new variants of old foes and by new diseases. Modern agricultural intensification practices have heightened the challenge - the planting of vast swathes of genetically uniform crops, guarded by one or two inbred resistance genes, and use of single target site antifungals has hastened emergence of new virulent and fungicide-resistant strains. Climate change compounds the saga as we see altered disease demographics - pathogens are on the move poleward in a warming world.

This presentation will highlight some current notable and persistent fungal diseases. It will highlight the evolutionary drivers underpinning emergence of new diseases and allude to the accelerators of spread. I will set these points in the context of recent disease modelling, which shows the global distributions of crop pathogens and their predicted movement and will discuss the concept of crop disease saturation.

I shall conclude with some thoughts on future threats and challenges, on fungal disease mitigation and of ways of enhancing global food security.

Fisher, M, Henk, D, Briggs, C, Brownstein, J, Madoff, L, McCraw, S and Gurr, S.J. (2012) "Emerging fungal threats to animal, plant and ecosystem health" *Nature*, **484**:185

Bebber, D.P., Ramotowski, M.A.T., Gurr, S.J. (2013). Crop pests and pathogens move poleward in a warming world. *Nature Climate Change*, **3**, 985

Bebber, D., Holmes and Gurr S.J (2014) The global distribution of crop pests and pathogens. *Global Ecology and Biogeography* 23:1398

Bebber, D.P., Holmes, T., Smith, D and Gurr S.J (2014) Economic & physical determinants of global distributions of crop pests & pathogens. *New Phytologist* 202.901

Bagchi, R., Gallery, R.E., Gripenberg, S., Gurr, S.J., Narayan, N., Freckleton, R.P., and Lewis, O.T. (2014) Pathogens and insect herbivores drive rainforest plant diversity and composition. *Nature* **506**:85

Bebber, D and Gurr.S.J (2015) Crop-destroying fungi and oomycetes challenge global food security. **Fungal Genetics and Biology** **74**: 62

Fones, H. and Gurr, S.J (2015) The impact of Septoria tritici blotch disease on wheat: an EU perspective. **Fungal Genetics and Biology** **79**: 3

Falloon, P., et al., (2015) Using climate information to support crop breeding decisions and adaptation in agriculture. **World Agriculture** **5** 25

Bebber, D., Delgado, A and Gurr, S.J. (2016) Modelling fungal pathogen infection risk with climate reanalysis data. **Proc Royal Soc B** 371 1709 20150458

Fisher, M.C., Gow, N.A.R and Gurr, S.J. (2016). Tackling emerging fungal threats to animal health, food security and ecosystem resilience
**Proc Royal Soc B** 371 1709 20160332

Fones, H.M., Mardon, C.G., and Gurr, S.J. (2016) A role for asexual spores in infection of *Fraxinus excelsior* by the ash-dieback fungus *Hymenoscyphus fraxineus*. *Scientific Reports*, *Nature Journals* **6**

**337W Role of plant-endophyte interactions in shrub encroachment.** M.I. Hutchinson, A.J. Robinson, D.O. Natvig Department of Biology, University of New Mexico, Albuquerque, NM.

Desertification is a significant global phenomenon, and in many dryland areas shrub encroachment is considered a primary factor. Shrub encroachment, the conversion of grasslands into shrublands, is a well-documented process but its causes are debated. Here, we
investigate how interactions between plants and fungal root endophytes may explain the expansion of shrubs into arid grasslands by examining the fungal colonization of roots from two Chihuahuan desert plants, the C3 shrub *Larrea tridentata* (creosote) and the C4 grass *Bouteloua eriopoda* (black grama). Our sampling spanned two years (2012 and 2015) and a narrow geographical gradient at the Sevilleta National Wildlife Refuge, NM, where there is a distinct ecotone between shrubland and grassland. For each sampling period we collected roots from 10 creosote plants, 5 black grama plants within the creosote stand, and 5 black grama plants from the grassland. To assess fungal colonization, we sequenced the fungal ribosomal internal transcribed spacer (ITS) from both cultured endophyte isolates and from the total DNA of harvested roots via high-throughput sequencing. ITS sequences showed consistency across years but significant differences among the fungal communities of the three sample types (although grama samples were more similar to each other than to creosote). A majority of cultures from creosote roots and a portion of cultures from the transition-zone black grama roots were from the genus *Monosporascus*. No cultures from grassland black grama roots were represented by *Monosporascus*, although sequences from members of the genus were observed among 454 sequences. While previous studies demonstrated an aridland fungal endophyte community shared across diverse plant species at the Sevilleta, our results demonstrate that endophyte communities are also shaped by host species, supporting the possibility that endophytes influence encroachment.

**338T Intestinal mycobiome variation across geography and phylogeny in the snail genus Conus.** A. Quandt, A. Glasco, T. James Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI.

This project seeks to use the extensive collections of a hyper-diverse, predatory genus of marine snails, *Conus*, at the University of Michigan Museum of Zoology to fill a gap in our understanding of the fungi that associate with mollusks and how the mycobiome (fungal community) varies across geographic space, phylogenetic diversity, and ecological niches of the hosts. Several species of *Conus* (including *C. ebraeus*), have near global distributions and are particularly well represented in the collections, yet we have no knowledge of whether the mycobiome of such animals will be driven by their geographic location or their phylogenetic relationships. To determine mycobiome compositions, we dissected the intestines and digestive gland tissues from more than 30 *Conus* spp. from over 20 different countries, extracted DNA, and used fungal-specific amplicon-based high throughput sequencing of the ribosomal DNA region. First, we identify which fungi are commonly and uniquely associated with the digestive tracts of *Conus* spp., all of which are dominated by *Malassezia* spp. We also explore whether *Conus* phylogeny or geography causes greater variation in fungal community structure, and discuss techniques for targeted sequencing of preserved museum specimens for studies.

**339F Plant-Microbe Interfaces: Characterizing the diversity and function of the ectomycorrhizome of Populus trichocarpa.** J.Alejandro. Rojas¹, Brian Looney²,³, Sunny Liao¹, Jay Chen², Jessy Labbé², Rytas Vilgalys¹ 1) Biology, Duke university, Durham, NC; 2) Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN; 3) University of Tennessee, Knoxville, TN.

The *Populus* root microbiome harbors a diverse community of ectomycorrhizal fungi (EMF) that significantly increase nutrient uptake and acquisition by the plant host while also providing protection against antagonistic parasites. Over 30 genera of EMF are known to associate with *Populus*, including many groups of mushroom-producing families including Boletaceae, Russulaceae, Cortinariaceae, Tricholomataceae, and Amanitaceae. A major aim of the PMI project is the collection, isolation, and characterization the diversity and function of major EMF fungal associates of *P. trichocarpa* across its range in the Pacific Northwest. In Autumn 2015 and 2016 (Oct/Nov 2015 and 2016) we surveyed macrofungal diversity under native *P. trichocarpa* forests from five core watersheds on the Squamish (BC), Snohomish (WA), Puyallup (WA), Columbia (OR and WA), and Willamette (OR) rivers. This resulted in over 150 collections of EMF fruit body collections and sampling of bulk soil used in bioassay studies. All sporocarp collections were plated on modified Melin-Norkrans medium, photographed, spore printed, and dried for identification and accession into fungal herbaria. The first fungal collections have been identified while the second samples from the second campaign are being identified by a consortium of taxonomic experts using the ITS barcode marker as well as morphological features.

**340W Transcriptome analysis of deep-sea fungi from the Gulf of Mexico.** P. Velez, R. Riquelme Departamento de Microbiología, Centro de Investigación Científica y de Educación Superior de Ensenada, Ensenada, Baja California Norte, MX.

The Gulf of Mexico is a biodiverse ecosystem that provides a wide array of valuable resources. Particularly, petroleum exploitation in this region sustains a multi-millionaire industry. However, these operations have resulted in major environmental disasters of unknown ecological consequences. Understanding the effect that oil spills have on marine ecosystems is limited to the extent of our knowledge on the autochthonous biota. Currently a great amount of information is available on bacteria inhabiting deep-sea sediments, however the occurrence of fungi in this environment has been poorly studied. Fungi are involved in key ecological deep-sea processes, yet these microorganisms have been rarely cultured and preserved from deep-sea samples. Therefore, the aim of this work is to analyze the microfungal diversity isolated from deep-sea sediment samples from the Gulf of Mexico, and to evaluate their metabolic response when exposed to hydrocarbons. We chose a culture-based approach in order to obtain fungal isolates, which will provide the opportunity for a *posteriori* studies and the possibility of *ex situ* preservation of fungal genetic resources. Fungal taxa were identified based on the evaluation of sequence data from the nuclear ribosomal internal transcribed spacer including the 5.8 rDNA region. Our results agree with previous work on deep-sea fungi reporting low levels of cultivable diversity, with Ascomycota as the dominant phylum. We did not observe significant differences among several culture media tested, indicating that perhaps these taxa tolerate a wide range of nutrient conditions. Ongoing work focuses on the transcriptome analysis on selected deep-sea fungal isolates, in order to provide the first insight of the genetic expression in response to hexadecane and 1-hexadecene. This is the first analysis exploring cultivable fungal diversity from deep-sea sediments in the Gulf of Mexico, and their response to hydrocarbons input, shedding light on a better understanding of the deep-sea ecosystem dynamics and bioremediation using deep-sea native taxa.

**341T The exciting content in your cup of tea.** I. Skaar¹, Y Zhang², M Sulyok⁴, X Liu⁵, M Rao⁶, J Taylor³ 1) Mycology, Norwegian Veterinary Institute, Oslo, NO; 2) School of Life Sciences, Shanxi University, Taiyuan, Shanxi, China; 3) Department of Plant and Microbial Biology, University of California, Berkeley, California, USA; 4) Center for Analytical Chemistry, Department of Agrobiotechnology, University of Natural Resources and Life Sciences, Tulln, Austria; 5) State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China and; 6) Bureau of Culture and Sports, Puer, Yunnan, China.
Tea is one of the most popular and widely consumed beverages in the world. Tea has important physiological effects on consumers due to the presence of compounds such as polyphenols, amino acids, vitamins, carbohydrates, caffeine, and purine alkaloids, all of which can have health benefits On the other hand, tea may also offer a complex microbiological diversity which can both contribute to the terroir of the tea, but also lead to presence of adverse secondary metabolites. Some teas are produced by microbiological fermentation. An example of such teas is the Pu-erh tea, produced in Yunnan, China, by fermentation of fresh *Camellia sinensis* leaves. The production and quality of Pu-erh tea is consequently closely related to microbial activity, making it important to understand the Pu-erh microbiome. The fungal diversity in teas and the corresponding fungal metabolites as revealed both by culture dependent and culture-independent approaches will be discussed.

We will present a study of fungal and bacterial communities leaves and two types of Pu-erh teas by high-throughput, rDNA-amplicon sequencing and by quantitative liquid chromatography-tandem mass spectrometry of their metabolites.

We identified 390 fungal and 629 bacterial OTUs from leaves and both Pu-erhs. Major findings were: 1) fungal diversity drops and bacterial diversity rises due to raw or ripened fermentation, 2) fungal and bacterial community composition changes significantly between fresh leaves and both raw and ripened Pu-erh, 3) aging causes significant changes in the microbial community of raw, but not ripened, Pu-erh, and, 4) ripened and well-aged raw, but not young, Pu-erh have similar microbial communities. Twenty-five toxic metabolites, mainly of fungal origin, were detected, with patulin and asperglaucide dominating. We show that principles of community ecology can be applied to microbially fermented foods, that the acceleration of Pu-erh fermentation by the ripened process approximates the slower, traditional process, and that discarding the first preparation of Pu-erh tea and saving the wetted tea to brew a second cup for consumption has a sound mycotoxicological basis.

**342F A phylogenetic approach based on PCR target enrichment and high-throughput sequencing for fungi: The *Ilyonectria* complex.** *J.Alejandro. Rojas*, Khalid Hameed, Rytas Vilgalys *Biology*, Duke university, Durham, NC.

The use of high throughput sequencing (HTS) has facilitated the study of the systematics and phylogenetics of organisms. Nonetheless, non-model organisms and complex systems in fungi require large datasets to study the phylogenetic relationships among their members, which has been traditionally approach by sanger sequencing multiple genes to resolve their taxonomy. The use of HTS could provide large amounts data for multiple genes suitable for phylogenetics analyses. The present study used ten unlinked genes, eight nuclear genes (LSU, SSU, ITS, RPB1, RPB2, TUB, ACT and EF1-α) and two mitochondrial genes (ATP6 and mitSSU) that can be amplified from a single individual, using a barcoding approach per individual. We describe a novel approach using PacBio sequencing to obtain ~1kb sequences, using a multilocus sequence typing (MLST) approach and the downstream analysis to genotype 100 individuals in single sequencing run, providing enough loci to characterize a species complex. This approach was applied to *Ilyonectria* complex, an endophyte present in cottonwood (*Populus trichocarpa* and *Populus deltoides*), but also associated with black-foot disease on grapevines. The genus *Ilyonectria* was recently separated from other *Cylindrocarpon*-like anamorphs, and 17 species have been described up to now, most of them associated with disease. The HTS-MLST approach was used on endophytic and pathogenic isolates of *Ilyonectria* to resolve the species complex associated with *Populus*. The methodology could be applied to study the phylogenetics and systematics of other fungal groups.

**343W Species and trichothecene genotype of Fusarium head blight Pathogens in Nebraska.** *E. Valverde-Bogantes*¹, S. Wegulo², H. Hallen-Adams¹ 1) Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE; 2) Department of Plant Pathology, University of Nebraska-Lincoln, Lincoln, NE.

Fusarium head blight (FHB) is a devastating and economically important disease that affects wheat, barley, oats and other small grain cereals. FHB is particularly problematic because it poses a threat to both food security and food safety, due to yield reductions and mycotoxin contamination. FHB is caused by many *Fusarium* species, but *F. graminearum* is the most prevalent causal agent in North America. The most common mycotoxins produced are trichothecenes, mainly deoxynivalenol (DON), acetylated derivatives and nivalenol (NIV), which are harmful both to humans and animals. Early studies reported the 15-ADON genotype as the dominant genotype in North America. However, genetically divergent populations with the 3-ADON and NIV genotypes have been reported, making the introduction of these genotypes into Nebraska feasible and highlighting the need for increased vigilance. This research aims to identify the species and trichothecene genotype of FHB pathogens in Nebraska, in order to identify the major toxigenic risks in the state. A total of 33 affected wheat samples were collected from Nebraska fields – 23 from 2015 and 10 from 2016. DNA was extracted directly from plant material, as well as from single-spore isolates, in order to compare results and reduce testing time. A PCR reaction was performed using two alternative sets of primers for *F. graminearum* – galactose oxidase (GO) gene and UBC85 primers. Additionally, a multiplex PCR using two alternative sets of primers based on the *Tri3* and *Tri12* genes was used to determine trichothecene genotype. Preliminary results show that *F. graminearum* of the 15-ADON genotype is still the most prevalent FHB pathogen in Nebraska. Thus far, similar results have been obtained when using plant-extracted DNA and single spore isolate DNA, except for two single-spore isolates that produced unusual results with the trichothecene genotype primers. Since isolates with the 3-ADON and NIV genotype have been increasing in range due to selective advantages, constant surveillance is important for the deployment of proper management strategies and to ensure detection methods are appropriate.

**344T Variations of mutation in *AVR1* and *AVR2* genes in the field isolates of tomato wilt fungus, *Fusarium oxysporum* f. sp. *lycopersici*.** *T. Arie*¹,²,³,⁴,⁵, K. Akai¹,³, S. Tamura³, S. Kikuchi⁴, Y. Ayukawa⁵, Y. Akagi⁶, K. Komatsu²,³,⁵, M. Kodama⁶ 1) Graduate School of Agriculture, Tokyo University of Agriculture and Technology (TUAT); 2) Institute of Global Innovation Reserch, Tokyo University of Agriculture and Technology (TUAT); 3) Laboratory of Plant Pathology, Department of Agriculture, Tokyo University of Agriculture and Technology (TUAT); 4) Leading Graduate School, Tokyo University of Agriculture and Technology (TUAT); 5) United Graduate School of Agriculture, Tokyo University of Agriculture and Technology (TUAT); 6) United Graduate School of Agriculture, Tottori University.

Three races (race 1, 2 and 3), determined by the host-specificity to cultivars, have been reported in the tomato wilt pathogen, *Fusarium oxysporum* f. sp. *lycopersici* (*Fol*). Molecular mechanisms of divergence of races in *Fol* have been explained by holding and mutation of the avirulence genes *AVR1* and *AVR2*; race 1, *AVR1* *AVR2* *AVR3*; race 2, *avr1* *AVR2* *AVR3*; race 3, *avr1* *avr2* *AVR3*. We analyzed the patterns of mutation of avirulence genes in the field isolates of *Fol*. Five patterns of mutation were recognized in *AVR1*; *AVR1*-null, *AVR1*
is truncated at nts. 380 or 685 with a transposon *hormin* (*avr1*<sup>th380</sup> or *avr1*<sup>th685</sup>), or expression of *AVR1* is hindered by the insertion of a transposon *fot-5* at 380 bp. upstream of *AVR1* (*avr1*<sup>tf-380</sup>), or by truncation at 212 bp. upstream of *AVR1* (*avr1*<sup>tf-212</sup>). Six patterns of mutations were recognized in *AVR2*; nucleotide substitutions at nts. 121 (*avr2*<sup>G121A</sup>), 134(*avr2*<sup>G134A</sup>), 137(*avr2*<sup>G137C</sup>), 122(*avr2*<sup>T122A</sup>), and 146(*avr2*<sup>C146T</sup>), or *AVR2*-null. In total 4 biotypes of race 2 and 7 biotypes of race 3 were identified in the collection by holding and mutation pattern of *AVR1* and *AVR2*. Three biotypes of race 2 and five biotypes of race 3 were isolated after 2000 in Japan. We discuss the evolutional path of development of races in *Fol*.

**345F Vertical transmission of cryptic alleles in Aspergillus flavus.** R.A. Olarte, K.E. Bushley Plant and Microbial Biology, UMN, St. Paul, MN.

*Aspergillus flavus* commonly infects agricultural staple crops, but is also a common species in natural systems. It and other *Aspergillus* species produce aflatoxins and cyclopiazonic acid (CPA), both of which are mycotoxins and secondary metabolites. *A. flavus* is the most potent producer of aflatoxins, and along with *A. parasiticus* are considered to be the *Aspergillus* species of agronomic importance. Differing lineages of *A. flavus* may be prone to chromosomal instability in the aflatoxin gene cluster, evidenced by variation among strains within a lineage in their ability to produce aflatoxin or CPA. For example, *A. flavus* lineage 1B is prone to large-scale gene loss events within the aflatoxin cluster, which results in a non-functional aflatoxin gene cluster. However, the rates of chromosomal instability across the diverse lineages of *A. flavus* and their consequences for variation in aflatoxin production remain unknown. Through PCR, progeny isolates were found to possess genetic elements (i.e., cryptic alleles) that were not detected in either parental strain. Cryptic alleles are believed to be extra-chromosomal elements that may result from genomic instability because inheritance of these alleles occurs in a non-Mendelian fashion. We sequenced a set of isolates, including the two parental strains as well as several progeny isolates, with Illumina HiSeq 4000 with conditions that allowed us to recover a minimum genomic coverage of at least 100x. Deep sequencing will confirm whether or not cryptic alleles found in the progeny are also present in the chromosomal genomes of the parental strains. *De novo* genome assemblies will be produced for isolates harboring cryptic alleles to be able to locate the positions of the identified cryptic alleles in the genomic landscapes of these isolates. Future research will determine whether these cryptic alleles are associated with fitness advantages and/or consequences.

**346W The genome of an uncultured nematode-destroying fungus and its role in resolving the zygomycete tree of life.** K.R. Amses, T.Y. James Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI.

Phylogenomic analyses of the artificial Zygomycota *s.l.* show support for its reorganization into two segregate phyla, each containing three subphyla. Of these six subphyla of zygomycetes, many are represented by only one or a few draft genomes, leading to some ambiguity in analyses. The sampling of more taxa from these poorly represented groups will bolster the strength of phylogenomic hypotheses formed at these deep nodes, strengthening the lines delineating the proposed taxonomic groupings. The Zoopagomycotina, a subphylum comprised of obligate parasites of other zygomycetes and small soil animals, is represented by a single draft genome in the most recent analyses. Establishment of axenic cultures has proven a central obstacle to the genome-enabled study of these cryptic soil fungi as acquisition of sufficient material for traditional whole genome sequencing is difficult. Single cell genomics approaches offer a solution to these complications, allowing for the sequencing and assembly of whole genome data sets from minicube amounts of input material summing to only one or a few cells. Leveraging these techniques in order to address the underrepresentation of Zoopagomycotina in phylogenomic analyses, we have sequenced the genome of *Stylopage hadra*, a cryptic but ubiquitous fungus that captures and consumes nematodes in natural and agricultural soils. Our single cell genomics approach generated four sequencing libraries with an average cumulative scaffold size of 22.81 Mbp and genome completeness scores of 56.65%, suggesting a *S. hadra* genome size of approximately 40 Mbp. Preliminary phylogenetic analyses based on concatenated RPB1 and RPB2 sequences support *S. hadra's* placement in the Zoopagomycotina. Sequencing libraries contain a putative endosymbiont of *S. hadra* belonging to the beta-proteobacterian genus *Glomeribacter* with cumulative scaffold sizes indicating a genome size of approximately 1.80 Mbp. Known from the cytoplasm of arbuscular mycorrhizal fungi and species of *Mortierella*, species of *Glomeribacter* comprise a group of opportunistic soil-borne endosymbionts that tend to reduce the growth rates of their associated fungi, indicating a weakly parasitic interaction.

**347T Genomics and sampling of the Ustilaginomycotina subphylum.** S.J. Mondo¹, T. Kijpornyongpan², M.C. Aime², I.V. Grigoriev¹ 1) Fungal Genomics, Joint Genome Institute, Walnut Creek, CA; 2) Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN.

Despite their importance as plant and animal pathogens, as a whole Ustilaginomycotina remains one of the most poorly understood lineages of Basidiomycota. As genomes are becoming available for more orders, comparative genomics is becoming an attractive tool for further characterization of this subphylum. At the JGI, we now have annotated genomes available for 16 Ustilaginomycotina, representing 9 different orders. Most of these genomes are small (ranging 8 to 27 Mb) and very compact. However, this sampling still represents a small subset of the total diversity present within Ustilaginomycotina. Here we discuss the sequencing progress and preliminary results comparing these genomes to one another and those from other fungal groups. Through collaborations with the Ustilaginomycotina community, our aim is to provide a comprehensive set of genomes to facilitate deeper exploration of this subphylum.

**348F First report of Rhizoctonia solani AG-4 on tomato in Pothwar region of Pakistan.** A.S. Gondal¹, A. Rauf², G. Irshad³, F. Naz⁴ 1) Department of Plant Pathology, PMAS Arid Agriculture University Rawalpindi, Rawalpindi, Punjab, PK; 2) Department of Plant Pathology, PMAS Arid Agriculture University Rawalpindi, Rawalpindi, Punjab, PK; 3) Department of Plant Pathology, PMAS Arid Agriculture University Rawalpindi, Rawalpindi, Punjab, PK; 4) Department of Plant Pathology, PMAS Arid Agriculture University Rawalpindi, Rawalpindi, Punjab, PK.

*Rhizoctonia solani* Kühn (teleomorph = *Thanatephorus cucumeris* (Frank) Donk) is the most important soil-borne fungal pathogen cause causes significant establishment with typical symptoms of collar rot, seedling death, stunted growth and root rot in tomato. Fungus isolated from diseased plants was identified as *Rhizoctonia solani* by comparing its morphological characters. Isolates were further confirmed as AG-4 by hyphal fusion test with tester isolates. Present study is the first report of *R. solani* AG-4 effecting tomato in Pothwar region, Pakistan.
**349W** Whole genome sequence of *Endogone lactiflua* reveals signatures associated with ectomycorrhizal lifestyle. *Y. Chang*¹,
A. Desirò², G. Bonito², J. Spatafora¹ 1) Botany and Plant Pathology, Oregon State University, Corvallis, OR; 2) Plant Soil and Microbial
Sciences, Michigan State University, MI.

*Endogone* (Endogonales, Mucoromycotina) is a species-poor but globally distributed genus and is known primarily from its truffle-like sporocarps. Ecologically *Endogone* includes saprobic to ectomycorrhizal (ECM) species and represents an independent origin of ECM outside Dikaryon. Together with its relatives in the early-diverging fungal lineage of Mucoromycotina, *Endogone* plays an important role in our understanding of the evolution of various plant-fungus associations and the molecular basis for these associations, especially for ECM.

In this study we did shotgun sequencing of the ECM species *E. lactiflua* from sporocarp tissue. We binned the sequence data using both
Blast-based and nucleotide-composition-based methods before final assembly and annotation. We identified a large number of
Mycoplasma-related endobacteria (MRE) sequences during the binning process. This is consistent with the observation that many
*Endogone* species harbor MREs. The genome size of *E. lactiflua* is much larger than the average fungal genome, with an estimation of ~
180 MB. Its large size is primarily due to the presence of various repetitive elements. Compared to its saprobic relatives, *E. lactiflua* has low
diversity and low copy numbers of genes coding Carbohydrate-Active Enzymes (CAZymes) in its genome. This is consistent with the
hypothesis that ECM lifestyle requires a small number of plant-degrading enzymes to avoid damaging host plant cells and eliciting a host
plant defense response.

**350T** Appalachia Fungal Diversity, their Importance in Industry Representation, and the Phenotypic Variation of Newly Isolated Strains. *Matthew Winans* Biology, West Virginia University, 53 campus drive, Morgantown, WV, 26505.

*Saccharomyces cerevisiae* is one of the most powerful tools for the genomic fungal scientific community. The genomic sequences of many *Saccharomyces* clade members show nucleotide divergence similar to the divergence between humans and birds. Lab strains are typically used for genomic analysis for their flexibility in the laboratory setting, which in many cases reduces genetic and phenotypic diversity. Typically, lab strains are used to account for diversity in biofilms and flocculation abilities which is important the medical research setting, accounting for $148B$ per year and in addition in the brewing industry which accounts for $220B$ per year in the US alone. Isolating new strains of *Saccharomyces* clade members makes it possible to gain a larger picture of the genetic variation present in the wild and determine how they are evolutionarily adapted to their environments. Assays to determine chemical tolerances of strains in response to glyphosate, copper, and copper nanoparticles were performed. Additionally, in this study, over 300 environmental samples were collected, isolated, and analyzed through optimization of isolation techniques, from trees, flowers, fruit, soil and mammalians in Appalachia, Mexico, and Chile at varying temperatures. These findings have shown great ubiquity not only in the *Saccharomyces* clade, but of yeasts in general and their unique phenotypes.

**351F** Using intra-species fungal diversity to get different epigenetic imprints on fungal exometabolite production. *J. Frisvad* CMB, Dept Systems Biol - DTU, Kongens Lyngby, DK.

Fungi that are primarily Competition-selected (C-selected), such as species of *Aspergillus*, *Penicillium Paecilomyces* and *Talaromyces*, have been reported to produce many different secondary metabolites (exometabolites). Some of these exometabolites are produced on commonly used agar or broth media, while others need to be epigenetically induced by exometabolites from other species. Alternatively histone de-methylation or deacetylation inhibitors may be added to a medium in order to increase the number and kinds of exometabolites produced by the fungus. Often the use of an additional medium will increase the number of members of one biosynthetic family of exometabolites. On the commonly used media Czapek yeast autolysate (CYA) agar and yeast extract sucrose (YES) agar *Aspergillus taichungensis* did not produce any prenylated indol alkaloids, while on rice the same isolate produced 21 different taichunamides. However exometabolites coded by apparently silent gene clusters can also be discovered by examining isolates of the same species from different geographic regions. *Penicillium antarcticum* is a marine-derived fungal species producing a series of bioactive exometabolites. 47 strains from all over the world were analyzed using HPLC-DAD after growth on CYA and YES agar. Of these 45 produced asperentins, 40 patulin, 31 antarones, 28 fischerin, 25 atlantinone A, 23 chrysogines, 22 phthalides, 12 penitrems, 12 deacetoxyfructigenine, 5 a terrestric acid-like metabolite, 4 atrovenetins, 4 austalides, 3 cytochalasins, 1 an orthosporin-like metabolite and 1 patulodins. Furthermore potentially new extrolites (chromophore families not observed in any other *Penicillium* species) were produced by 26, 11, 5, 4, 3, 2, and 6 singleton isolates respectively. Examining several isolates within a species from different niches and geographic regions is an alternative way of discovering potentially bioactive exometabolites that could otherwise only be discovered by genome sequencing and using exometabolite gene cluster search algorithms. These observations indicate that Aspergilli and Penicillia can be epigenetically imprinted to express their exometabolites in certain instances.

**352W** Genetic diversity of *Ustilago esculenta*, a smut fungus associated with water bamboo in East Asia. *W.C. Shen* Plant Pathology & Microbiology, National Taiwan Univ, Taipei, TW.

*Ustilago esculenta* Henning is a smut fungus associated with a wild rice species, *Zizania latifolia* Turcz. With the infection of this smut fungus, swollen gall is developed at the basal part of the plant and becomes a favorable vegetable, so called water bamboo or makomotake, in East Asia. Sexual differentiation is required for the establishment of fungus-plant association and development of edible galls is resulted from phytohormone imbalance. In this study, we aim to characterize the mating type loci and mating system of *U. esculenta* and determine its genetic diversity. *U. esculenta* isolates were first collected from teliospores and gall tissues of field materials in Taiwan and Japan extensively. To identify the mating type loci and idiomorphic polymorphism, we conducted NGS genomic and PCR screening approaches. Conservations of pheromone, pheromone receptor and homeodomain transcriptional factors were confirmed and three idiomorphs are identified. Interestingly, transposon was found to insert at the mating type locus of *U. esculenta*. Genetic evidence indicate that *U. esculenta* contained a bipolar heterothallic mating system involved extremely large mating type loci. To understand its genetic diversity, we have targeted transposon and simple sequence repeats in *U. esculenta* genome and tried to develop molecular markers for fingerprinting study. These information will be utilized to breed for new varieties for commercial production.
**353T Microbial communities associated with external fur surfaces of bats and the discovery of novel isolates with antifungal activity against *Pseudogymnoascus destructans*.** Paris Hamm¹, Shiloh Lueschow², Ara Winter³, Nicole Caimi³, Diana Northup³, Ernest Valdez⁴, Debbie Buecher⁵, Christopher Dunlap², David Labeda⁶, Andrea Porras-Alfaro¹ ¹) Department of Biological Sciences, Western Illinois University, Macomb, IL; 2) Crop Bioprotection Research Unit, US Department of Agriculture, Peoria, IL; 3) Department of Biology, University of New Mexico, Albuquerque, NM; 4) United States Geological Survey, Fort Collins Science Center, Albuquerque, NM; 5) Buecher Biological Consulting, Tucson, AZ; 6) Mycotoxin Prevention and Applied Microbiology Research Unit, US Department of Agriculture, Peoria, IL.

White-nose Syndrome (WNS), a bat fungal disease caused by the psychrophilic fungus *Pseudogymnoascus destructans*, has been estimated to have caused the death of more than six million bats in the eastern U.S. and Canada. Fungal and bacterial surveys were conducted to explore bats' natural microbial communities as a possible defense against this pathogen. Our WNS research efforts have mainly focused on the study of microbial communities associated with bats using next generation sequencing, metagenomic analysis, and the evaluation of cultured Actinobacteria to determine antifungal activity against *P. destructans*. Metagenomic analysis showed that fungi on bat fur surfaces belong to Chytridiomycota, Ascomycota, Basidiomycota, Glomeromycota, and Neocallimastigomycota. Distinct fungal and bacteria communities were detected on WNS (+) bats from Illinois and Indiana and WNS (-) bats from New Mexico and Arizona using 454 pyrosequencing. Further, we evaluated the antifungal potential of naturally occurring Actinobacteria isolated from WNS (-) bats. Bacteria colonizing bat fur and membranes were isolated from 12 healthy bat species providing approximately 2,700 isolates. We have screened over 1,300 bacterial isolates using a bi-layer method, of which 36 isolates show antifungal activity against *P. destructans*. Of the antifungal producing Actinobacteria, 32 (89%) were from the genus *Streptomyces*, known for their antibiotic production. Fifteen of the isolates with antifungal activity against *P. destructans* were identified as novel *Streptomyces* species after morphological and multi-gene phylogenetic analysis. This study includes one of the largest surveys of microbial communities associated with healthy bats. Our results show that bats in western North America possess novel bacterial microbiota with the potential to inhibit *P. destructans*.

**354F Elucidating fungal endosymbiont interaction mechanisms between *Mortierella elongata* and *Mycoavidus cysteinexigens*.** Jessie Uehling¹, Fred Dietrich¹, Pawel Misztal², Gregory Bonito³, Tim Tschaplinski⁴, Allen Goldstein², Chris Schadt⁴, Jessy Labbe⁴, Rytas Vilgalys¹ ¹) Genetics & Genomics, Duke University, Durham NC 27708, USA; 2) University of California at Berkeley, Berkeley, CA 94270 USA; 3) Michigan State University, East Lansing, MI 48823 USA; 4) Biosciences Division, Oak Ridge National Laboratory, PO Box 2008 MS 6445, Oak Ridge, TN 37831-6445, USA.

Recently efforts to catalog beneficial microbes associated with *Populus* have yielded cultures of diverse fungi including several *Mortierella elongata* (*Mortierellomycotina*) isolates. Some of these fungi including *M. elongata* strain AG77 harbor bacterial endosymbionts belonging to the Burkholderiales. We have sequenced the genome of *M. elongata* AG77 and its bacterial endosymbiont *Mycoavidus cysteinexigens* (AG77), and used comparative phylogenomics to infer this symbiosis is ancient and grounded in shared metabolites. We have used and developed methods to assemble single genomes from meta-genomic data and inferred several gene losses that make culturing endosymbiont *M. cysteinexigens* (AG77) challenging. To circumvent this research obstacle, we used an antibiotic treatment to create fungal strains with and without their long-term endosymbionts for comparative multi-omic studies. The ability to contrast cleared *M. elongata* strains and their wild type, endosymbiont-housing relatives in the same genetic background allows us to ask the following questions: 1. *How does the presence or absence of long-term, co-evolved endosymbionts impact health and physiology of host fungi?* 2. *Which currently available methods can be used and modified to study interaction dynamics for systems involving multiple intractable microbes?* Our research goals through this work were to develop omics and imaging pipelines that allow the study of bacterial fungal interaction mechanisms. To this end we have used genome sequencing, transcriptomics, metabolomics, and volomics for studying fungal endosymbiont interaction dynamics. The results of these experiments will be further discussed.

**355W Diversity of fungal communities and co-occurrence with bacterial communities at the MSU-KBS Long Term Ecological Research wheat/corn/soybean crop rotation.** Kristi Gdanetz MacCready, Frances Trail Michigan State University, East Lansing, MI.

The recent launch of the Phytobiomes Alliance calls for a holistic approach to studying plant health, one aspect of this is the plant microbiome. We need a thorough understanding of the populations of microbes inhabiting different plant niches before we can successfully manipulate microbial communities to benefit our crop health. Michigan State University's Kellogg Biological Station Long Term Ecological Research site harbors six replicate plots of four management treatments: conventional management, no-till, reduced inputs, and organic. These plots are on a three-year wheat/corn/soybean rotation cycle, providing an ideal site to conduct long-term characterization of these economically important row crops. We compared the diversity of the microbial communities, using fungal ITS2 and bacterial 16S amplicon sequencing, across the four management styles and three different plant organs for one cycle of the crop rotation. We assessed how below-ground and above-ground microbial communities differed, identified the unique community members for each crop, as well as the differences between the four management styles. Microbe communities, across plant organs and within a crop, are similar at the early vegetative stages; at later growth stages, the diversity increases. Compared with fungal communities, greater variation was observed in the structure of bacterial communities between organs and crops. Host plant species and plant organs had a stronger influence on community structure compared to plot management. Increased microbe diversity with time might be explained by a pattern of succession, or response to changes in plant metabolites and biochemistry across growth stages. Manipulating microbial communities to increase diversity, during critical growth stages when pathogens infect, may be beneficial to crop health.

**356T Genotypic analysis of *Rhodotorula* strains isolated from environmental and clinical samples.** Alex Metzger¹, Natalie Akers², Abigail Jacobs¹, Lauren McKean², *Amy Reese*¹ ¹) Basic Sciences, St. Louis College of Pharmacy, St. Louis, MO; 2) Biological Sciences, Cedar Crest College, Allentown, PA.

*Rhodotorula* species are pink to coral encapsulated basidiomycete yeasts that have been isolated from various environmental locations. They are also increasingly linked with disease in immunocompromised patients and are particularly associated with central venous catheter infections and meningitis. Little is known about the biology of these fungi, including their natural reservoirs and the role of their capsule. Clinical isolates are known to be naturally resistant to fluconazole, voriconazole, and caspofungin, leaving few antifungals available for treatment. We have collected environmental *Rhodotorula* species from trees, domesticated dog coats, and avian excreta. In
addition, we have obtained agricultural strains from a plant pathology lab and clinical isolates from a medical laboratory, resulting in a set of about 50 strains in total. To characterize and compare our strains we have grown them on various cell wall integrity-challenging media and at various temperatures, assessed urease production, performed capsule size analysis, and detected their ability to bind of our anti-capsular antibody. In order to correlate our phenotypic and biochemical data with genotypic information, the internal transcribed spacer (ITS) regions of eight environmental and eight clinical strains were amplified with ITS1 and ITS2 primers from genomic DNA and the amplicons sequenced directly with the same primers. These results verified that all strains were *Rhodotorula*, but the data were not ideal for species-level identification. We have extracted genomic DNA from all samples to amplify longer ITS regions with ITS1 and ITS4 primers to then subclone into plasmids for improved sequencing reads for detailed species-level identification. There is limited genetic data available for these fungi in fungal databanks and the methods used to determine the species of clinical isolates is often vague or missing in the literature. Phenotypic and genotypic analyses for *Rhodotorula* species would allow a better understanding of these fungi and could prove instructive as more infections arise in our immunocompromised patient population from this emerging pathogen.

## Gene Regulation

**357F A novel phosphoregulatory switch controls the activity and function of the major catalytic subunit of protein kinase A in Aspergillus fumigatus.** E.K. Shwab¹, P.R. Juvvadi¹, G. Waitt², E.J. Soderblom², M.A. Moseley², N.L. Nicely³, Y. Asfaw⁴, W.J. Steinbach¹,⁵ 1) Department of Pediatrics, Division of Pediatric Infectious Diseases, Duke University Medical Center, Durham NC, USA ; 2) Duke Proteomics and Metabolomics Core Facility, Center for Genomic and Computational Biology, Duke University, Durham NC, USA ; 3) Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC, USA; 4) Department of Laboratory Animal Resources, Duke University Medical Center, Durham NC, USA; 5) Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham NC, USA.

Invasive aspergillosis, caused by the filamentous fungal pathogen *Aspergillus fumigatus*, is a major cause of death among immunocompromised patients. The cyclic-AMP/protein kinase A (PKA) signaling pathway is essential for hyphal growth and virulence of *A. fumigatus*, but the mechanisms of PKA regulation remain largely unknown. Here we have discovered a novel mechanism for the regulation of PKA activity in *A. fumigatus* via phosphorylation of key residues within the major catalytic subunit, PkaC1. Phosphopeptide enrichment and tandem mass spectrometry revealed the phosphorylation of PkaC1 at four sites (S175, T331, T333 and T337) with implications for important and diverse roles in the regulation of *A. fumigatus* PKA. While the phosphorylation at one of the residues (T333) is conserved in other species, the identification of three other residues represents previously unknown PKA phosphoregulation in *A. fumigatus*. Site-directed mutagenesis of the phosphorylated residues to mimic or prevent phosphorylation revealed dramatic effects on kinase activity, growth, conidiation, cell wall stress response, and virulence in both invertebrate and murine infection models. Three-dimensional structural modeling of *A. fumigatus* PkaC1 substantiated the positive or negative regulatory roles for specific residues. Suppression of PKA activity also led to downregulation of protein expression in an apparent novel negative feedback mechanism. Based on these findings, we propose a model in which PkaC1 phosphorylation both positively and negatively modulates its activity. This work paves the way for the future discovery of fungal-specific aspects of this key signaling network.

**358W Recognition for Calcineurin in Aspergillus fumigatus by CRISPR-Cas9 mutagenesis.** C. Zhang, X. Meng, L. Lu College of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu, CN.

Calcineurin (CaN) as a conserved protein phosphatase activated through the binding of $Ca^{2+}$-calmodulin, is a heterodimer composed of a regulatory (CnaB) and a catalytic (CnaA) subunit. In all eukaryotes, the calcineurin pathway is an important signaling cascade. In fungal pathogens, calcineurin is required for hyphal growth; stress responses and virulence and calcineurin inhibitors are suggested to be effective antifungals against the major invasive fungal infection. However, in mammals, calcineurin inhibitors are widely used immunosuppressive drugs, which limit therapeutic effectiveness as antifungals. Through gene deletion combined with complementation test, previous studies reported that calcineurin A embeds fungal-specific residues which are required for phosphorylation and virulence, indicating this specific region could be an effective antifungal target in filamentous fungal pathogen *Aspergillus fumigatus*. However, the *précised in situ* molecular characterization of calcineurin remains unknown. In this study, to carry out precise genome editing, we set up a highly efficient CRISPR mutagenesis system for exploring the function of calcineurin. Different from data obtained in model organism *A. nidulans*, we found that the *cnaA* null mutation can be significantly suppressed by adding calcium chelator EGTA or by deletion of the subunit of high affinity calcium channel *cchA* while this suppression only occurred in the presence of a calcineurin binding protein calcipressin CbpA. These results indicate that the hyphal growth defects caused by loss of calcineurin are primarily due to the influx of extracellular calcium via the high affinity calcium channel in *A. fumigatus*. Furthermore, through site-direct point mutation approaches, we found that previous identified the fungal-specific Serine Proline Rich Region (SPRR) located between two conserved the CnaB-binding helix and the CaM-binding domain are not required for hyphal growth since the in situ 4SA mutant (in situ point mutation 4SA containing S406A, S408A, S410A and S413A in Serine Proline Rich Region) has almost a normal colony phenotype, which is inconsistent to previous reported. In contrast, in situ point mutation W374G at the CnaB-binding helix domain caused severe hyphal growth defects, suggesting this domain is required for hyphal growth. Further verification for detail is ongoing.

**359T The Negative Cofactor 2 complex mediates azole resistance through transcriptional regulation of an efflux transporter and ergosterol biosynthesis in Aspergillus fumigatus.** Takanori Furukawa¹, Fabio Gsaller¹, Josie Parker², Steve Kelly², Scott Moye-Rowley³, Paul Bowyer², *Mike Bromley*¹ 1) Manchester Fungal Infection Group, University of Manchester, Manchester, UK; 2) Institute of Life Science, Swansea University Medical School, UK; 3) Department of Molecular Physiology and Biophysics, University of Iowa.

*A. fumigatus* is the most important airborne mould pathogen and allergen worldwide. Estimates suggest that over 3 million people have invasive or chronic infections that lead to in excess of 600,000 deaths every year. Very few drugs are available to treat the various forms of aspergillosis and we rely predominantly on the azole class of agents (Itraconazole, Voriconazole, Posaconazole and the recently licensed Isavuconazole). Resistance to the azoles is emerging. For individuals that are infected with a resistant isolate the mortality rate exceeds 88%. Therapy failure is in part attributed to delays in administering alternative therapies so methods to rapidly detect resistance is critical. While resistance in around 50% of clinical isolates has been linked to modification of the gene encoding the target of the azoles,
*cyp51A*, our understanding of what leads to resistance in the remaining strains is lacking. To examine the mechanistic basis of non-target driven azole resistance we have carried out a systematic evaluation of the role of transcription factors in drug tolerance. We have screened a library of 401 transcription factor *A. fumigatus* knockout strains and identified a cohort of factors governing azole resistance and sensitivity. Here we report on two CBF/NF-γ family transcription regulators, AFUB_029870 (NctA) and AFUB_045980 (NctB) where loss of function leads to azole resistance. The orthologues of these regulators in yeast, known respectively as BUR6 and NCB2, are subunits of a heterotrimeric transcriptional regulator called Negative Cofactor 2 (NC2). In keeping with the hypothesis that NctA and NctB are part of the same transcriptional regulatory complex, we show the transcriptional regulons governed by these two factors are essentially identical. Furthermore, reciprocal co-immunoprecipitation studies using S-tagged versions of the proteins indicate that they interact directly with each other.

With a view to understanding which genes are directly regulated by NctA, we performed genome-wide protein-DNA interaction analysis using ChIP-seq. Over 1500 NctA interacting loci were identified, including 13 genes which encode members of the ergosterol biosynthetic pathway, and the efflux transporter cdr1B. Eight of these sterol biosynthetic genes along with cdr1B are transcriptionally upregulated in our NctA null leading to elevated levels of ergosterol and Cdr1B protein. Evaluation of the clinical significance of these findings is under investigation.

**360F The opposing roles of SrbA and the HapX/CCAAT binding complex in the regulation of sterol biosynthesis and azole tolerance in Aspergillus fumigatus.** F. Gsaller¹, P. Hortschansky², T. Furukawa¹, P.D. Carr¹, B. Rash¹, J. Capilla³, C. Müller⁴, F. Bracher⁴, H. Haas⁵, A.A Brakhage²,⁶, M.J. Bromley¹ ¹) Division of Infection, Immunity & Respiratory Medicine, University of Manchester, Manchester, GB; ²) Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany; ³) Microbiology Unit, Medical School, Universitat Rovira i Virgili, Spain; ⁴) Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians University of Munich, Germany; ⁵) Division of Molecular Biology, Biocentre, Medical University of Innsbruck, Austria; ⁶) Institute for Microbiology, Friedrich Schiller University Jena, Jena, Germany.

Azole drugs selectively target fungal sterol biosynthesis and are critical to our antifungal therapeutic arsenal. Resistance to this class of agents in the major human mould pathogen *Aspergillus fumigatus* is reaching levels that suggest that they could be lost for clinical use. One family of pan-azole resistant isolates, characterized by the presence of a tandem repeat of at least 34 bases (TR34) within the promoter of sterol C14-demethylase encoding gene *cyp51A* dominates and is found globally. We demonstrate the transcriptional mechanisms underpinning TR34 mediated resistance showing the repeat is bound by both the sterol regulatory element binding protein SrbA, and the *CCAAT binding complex (CBC)*. We show that the CBC acts complementary to SrbA as a negative regulator of ergosterol biosynthesis and TR34 driven overexpression of *cyp51A* results from effective duplication of SrbA but not CBC binding sites leading us to suggest possible routes to tackle the problem of TR34 mediated resistance.

**361W Ascospore-specific gene expression analysis in Aspergillus nidulans.** Mi-Kyoung Lee¹, Jong-Hwa Kim², Jae-Hyuk Yu¹, *Kap-Hoon Han*² ¹) Department of Bacteriology, University of Wisconsin-Madison, WI, USA; ²) Department of Pharmaceutical Engineering, Woosuk University, Wanju, Jeonbuk, South Korea.

Developmental process and spore formation in a model filamentous fungus *Aspergillus nidulans* is environmentally and genetically regulated. Asexual spores or conidia differentiation is controlled by various orchestrated developmental pathways, including the *brlA* gene-mediated conidiophore and conidia morphogenesis. However, a few genes have been elucidated for playing an important role in sexual development and ascospore formation. The *nsdD*, *nsdC* and *veA* genes are well-known key regulators of sexual and asexual developmental processes. However, unlike conidia, physiological and genetic studies of ascospores are remained to be characterized. To know more about the ascospores biology, we performed RNA-seq analysis from *A. nidulans* conidia and ascospores RNA samples. Comparative analysis of transcription profiles of conidia and ascospores revealed many genes that are expressed differentially in both spores. Detailed investigation of the differentially expressed genes is in progress.

**362T Genetic interactions among G-protein coupled receptors GprH, I, and –M affect nutrient sensing, sexual development and secondary metabolite production in Aspergillus nidulans.** Laura Mellado¹, Jessica M. Lohmar², Ana M. Calvo², Gustavo Henrique Goldman¹ ¹) University of São Paulo, Ribeirão Preto, Brazil; ²) Department of Biological Sciences, Northern Illinois University, Dekalb, USA.

All living cells have the capacity to sense and respond to external signals, fundamental for cellular functions and survival. In eukaryotic signal transduction, the heterotrimeric G protein system plays a crucial role. In fungi, heterotrimeric G proteins (GPCRs) mediate responses to cell growth and division, mating, virulence and pathogenicity and the production of secondary metabolites. In spite of its biological importance, only a few have been functionally characterized. Our previous study in *Aspergillus nidulans* revealed the increased expression of a putative uncharacterized GPCR, *gprH*, during carbon starvation. We have demonstrated that GprH is a putative glucose and tryptophan receptor and also a sexual development repressor. We identified two other putative GPCRs, *gprI* and *gprM*, as additional elements of the carbon starvation response. Microarray hybridization analysis and qRT PCR experiments showed a *gprH*-dependent expression of *gprI* and *gprM* under carbon starvation. The presence or absence of each protein has an impact on spore germination and/ or hyphal proliferation, making cells able to respond to the presence or absence of glucose in the culture media especially post-carbon starvation. GprH and GprI also negatively regulate sexual development. *ΔgprH* and *ΔgprI* mutants showed an increase in cleistothecia production, and subsequently, a reduction in conidiospores formation under light conditions. This phenotype is still conserved in darkness for the *ΔgprH* mutant but not for the *ΔgprI*. These GPCRs might play an important role in secondary metabolite production. Our results show that the deletion of these genes affect the production of the sterigmatocystin toxin, and it is dependent on the VeA transcription factor and the exposure to light. Double mutant analysis showed genetic interaction among these genes affecting their individual phenotypes. In summary, we propose that GprH, GprI and GprM are nutrient receptors and they are involved in the germination, hyphal proliferation, sexual development and secondary metabolite production in *A. nidulans*. Intriguingly, there is crosstalkling among glucose starvation, sexual development and light influencing the GPCRs signaling that seems to be mediated by VeA.

Financial support: FAPESP and CNPq, Brazil
**363F Global Analysis of CreA Regulatory Network in Aspergillus nidulans.** Yingying CHEN¹, Liguo DONG¹, Md Ashiqul Alam², Fang Wang¹, Joan Kelly², Koon Ho Wong¹ 1) Faculty of Health Science, University of Macau, Macau, CN; 2) Department of Genetics and Evolution, School of Biological Science, The University of Adelaide.

Carbon metabolism is central to all living organisms governing many physiological processes. In *Aspergillus nidulans*, carbon metabolism is tightly controlled at the transcription level by a process known as carbon catabolite repression (CCR). CCR is mediated by a global transcriptional repressor called CreA. Despite decades of research on CreA, the full spectrum of CreA targets and how it globally regulates and coordinates expression of genes remain unclear. In this research, we applied two powerful approaches RNA-seq and ChIP-seq for a genome-wide study of CreA under repressing and derepressing conditions. We discovered that CreA binds to several thousands promoters in the genome. The bindings are found mainly at the nucleosome-depleted promoter regions and are enriched with GC-rich motifs along with many other motifs. The set of CreA direct targets include genes involved in biosynthetic process, transmembrane transport, response to stimulus, regulation of transcription, ion homeostasis and sexual reproduction. Some of these roles have been confirmed by functional tests. Interestingly, our analysis reveals that the CreA binding intensity, the number of CreA binding sites and additional motifs are associated with different sets of CreA target genes of different physiological functions. Moreover, our genome-wide ChIP-seq data also revealed that CreA constitutively binds to the target promoters at similar levels under both repression and de-repression conditions, even for genes whose expression is markedly increased (i.e. de-repressed) under the de-repression condition. This observation strongly indicates that CreA binding alone is not sufficient for CreA regulation, and in turn implies that post-translational modification(s) play(s) crucial role in CreA regulation. Transcription profiling analysis by RNAseq of wildtype and *creA*? mutant strains reveals that CreA not only acts as a transcription repressor, but also has a positive role for many target genes. Surprisingly, we found that expression of more than half of CreA bound genes are not significantly affected in the *creA*? strain. This suggests that either CreA pauses at many promoters pending modification for function or the activators responsible for activating those genes are not available for function under our experimental conditions. Taken together, our results reveal a comprehensive global CreA regulatory network at a whole-genome level and illuminate novel CreA regulating patterns and functions.

**364W Phosphoproteomic and transcriptomic analysis of PKA activity in the filamentous fungus Aspergillus nidulans.** L.F.C. Ribeiro¹, C. Chelius¹, K. Boppidi¹, S. Linconl², R. Srivastava², J. Kumar³, S.D. Harris³, M.R. Marten¹ 1) Chemical, Biochemical and Environmental Engineering Department, University of Maryland, Baltimore County (UMBC), Baltimore, MD, United States; 2) Chemical & Biomolecular Engineering Department, University of Connecticut, Storrs, CT, United States; 3) Center for Plant Science Innovation, University of Nebraska - Lincoln, Lincoln, NE, United States.

The ability to sense and respond to changing nutrient conditions is an essential function for all cells. After a fungus senses a change in its external nutrient environment, several internal processes occur in a coordinated fashion, including transcription of relevant transport and metabolic genes, ribosome biogenesis, up or down-regulation of translation machinery, and expression of proteins which direct morphogenesis. A major pathway involved with nutrient sensing and regulation of cell metabolism is activated by cAMP-dependent protein kinase A (PKA). While some PKA substrates have been characterized, these are not sufficient to explain the significant range of PKA activity in the cell. To better understand PKA systemic action in *Aspergillus nidulans*, we carried out phosphoproteomic and transcriptomic analysis of two *A. nidulans* strains: PKA+ (wild type) and PKA- (*DpkaA*). The data have allowed us to identify a number of proteins affected by PKA, many of which have not been previously associated with PKA. This approach also allowed us to identify specific phosphorylation sites which appear to mediate the impact of PKA. For example, CreA, a transcription factor responsible for carbon catabolite repression, was observed to be phosphorylated only in the wild type. Since CreA is responsible for repressing the transcription of cellulolytic enzymes, cellulolytic activity was quantified for both strains cultivated in rich medium with glucose, and the *DpkaA* strain yielded significantly higher (2.5x) cellulase activity. These results suggest PKA phosphorylates CreA (directly or indirectly) at an identified phosphorylation site. Additional data imply the lack of this phosphorylation site inhibits the repressor activity of CreA.

**365T CRISPR/dCas9-mediated transcriptional activation in Aspergillus nidulans.** Shuhui Guo, Chirag Jayantibhai Parsania, Liguo Dong, Chris Koon Ho Wong Faculty of Health Sciences, University of Macau, Macau SAR, China.

The well-known and powerful CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat and associated protein Cas9) system has been extensively applied for genome engineering in many organisms. The system can be modified for transcriptional activation or suppression of gene-of-interest by linking a mutated Cas9 that lost the nuclease function (called dCas9) with a transcriptional activation element (e.g. VP16, VP64, VPR and SAM) or a repression element (e.g. KRAB, SID), respectively. In this study, we aim to establish and optimize a CRISPR/dCas9 system for transcriptional activation in *A. nidulans*. We have generated a strain that expresses the dCas9-VP64 fusion protein and a gRNA expression plasmid carrying an internal fragment of the wA gene for targeting to the wA locus. An efficient strategy for introducing gRNA sequences to the expression plasmid has also been optimized. Similar to the CRISPR/Cas9 system, targeting of dCas9 to specific genomic regions is also mediated by sequence specific gRNAs to PAM sites. However, the location of PAM sites is critical and can greatly influence the degree of transcriptional activation and repression by the CRISPR/dCas9 system. Since many PAM sites can be found across a given promoter region, it is not immediately obvious which one would be ideal for activation/repression. Based on the well-established characteristics (e.g. nucleosome density, chromatin modifications, DNA bendability, sequence conservation, etc.) underlying transcription factors function, we have designed an approach to predict PAM sites suitable for the purpose. As a proof-of-principle, we have tested the CRISPR/dCas9 activation system on genes located within active (e.g. house-keeping genes) or silent heterochromatin (e.g. genes within secondary metabolite clusters) regions in *A. nidulans*. The success and the level of transcriptional activation were determined using RT-PCR. A "PAM site for activation" prediction program will be developed for public use. We believe that the CRISPR/dCas9 activation system is not only useful for artificial transcriptional activation, but also offer a novel method for activating cryptic secondary metabolite gene clusters in filamentous fungi.

**366F L-rhamnose induction and metabolism in Aspergillus niger.** C. Khosravi, Maria Victoria Aguilar-Pontes, Eline Majoor, Ronald De Vries CBS Fungal Biodiversity Centre, Utrecht, The Netherlands.

*Aspergillus* species are potent producers of enzymes involved in plant polysaccharide degradation. In nature, *Aspergillus* degrades the polysaccharides to monomeric sugars that can serve as a carbon source. Therefore, *Aspergillus* uses a variety of catabolic pathways to
efficiently convert all the monomeric components of plant biomass.

L-rhamnose catabolism in fungi has been described for the yeasts *Pichia sptititis* and *Debaryomyces hansenii* (Watanabe et al., 2008). In *Aspergillus niger* this pathway is poorly studied, and none of the putative L-rhamnose pathway genes or enzymes have been characterized. This project aims to confirm the function of the putative L-rhamnose pathway genes and to identify which metabolite in the L-rhamnose pathway is the inducer of the L-rhamnose responsive regulator (RhaR) (Gruben et al., 2014). After identification of the candidates, deletion mutants for these genes were obtained. Growth profile result showed no or only minor growth for all of the metabolic mutants. To study the effect of deletion of these genes in more detail, transcriptomics analysis of the reference strain and the KO strains Δ*IraA*, Δ*IraB*, Δ*IraC* and Δ*rhaR* have been done. The results indicate that L-rhamnose, L-rhamnono-Y-lactone and L-rhamnonate are not the inducers of RhaR. Further experiments will be required in order to determine if the inducer is located at the end of the metabolic pathway.

**367W Subcellular localization and stability of deubiquitinase CreB involved in carbon catabolite repression in Aspergillus oryzae.** S. Ichinose, M. Tanaka, T. Shintani, K. Gomi Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan.

*Aspergillus oryzae* produces large amounts of amylolytic enzymes in the presence of maltooligosaccharides, In the presence of glucose, however, their production is repressed by carbon catabolite repression (CCR) system. In filamentous fungi, it has been proposed that CCR is regulated by Cys₂His₂ type transcriptional factor CreA, and creA gene deletion results in high production level of α-amylase in *A. oryzae* (1). In addition, it is known that the ubiquitin processing protease CreB is also involved in CCR regulation. We generated a *creB* deletion mutant in *A. oryzae*, and revealed that CCR was relieved by deleting the *creB* gene. However, it is not clear how CreB is involved in CCR regulation. In this study, to elucidate the mechanism of CCR regulation by CreB, we generated GFP- or 3FLAG-fused CreB and observed the subcellular localization and stability of CreB. CreB-GFP was localized in the cytoplasm in both glucose and maltose media. In contrast, the amount of CreB-3FLAG in maltose medium was larger than that in glucose medium. Concomitantly, the *creB* gene expression level in maltose medium was much higher than that in glucose medium. These results suggested that CreB is regulated at transcriptional level and the *creB* gene is induced under CCR relieved condition. In *Aspergillus nidulans*, it was reported that CreB interacts with WD40 repeat protein CreC. Thus, we generated a *creC* deletion mutant in *A. oryzae*, and observed that CCR was also relieved by deleting the *creC* gene in *A. oryzae*. Interestingly, *creC* gene deletion resulted in a significant decrease in the amount of CreB-3FLAG and increase in the *creB* gene expression level compared with the wild-type, while the subcellular localization of CreB was not substantially altered. These results suggested that stability and transcriptional level of CreB was regulated by CreC (1) Ichinose et al., *Appl. Microbiol. Biotechnol.*, **98**, 335–343 (2014)

**368T Development of genome editing method by direct introduction of Cas9-protein in Aspergillus oryzae.** K. Shimamoto¹,², R. Saitou¹, Y. Wada³, O. Mizutani¹, K. Oda¹, M. Okuda¹,², K. Iwashita¹,² 1) Brewing mycology, National Research Institute of brewing, Higashihiroshima, Hiroshima, JP; 2) Hiroshima Univ; 3) FASMAC Co.Ltd.

*Aspergillus oryzae* is the one of attractive industrial strains used for traditional fermentation industries and recent enzyme production and pharmaceutical industries. Molecular breeding of *A. oryzae* is still difficult and troublesome, because of its genetical difficulty such as multi-nuclear and deficient of cross. Recently, genome editing with CRISPR/Cas9 is focused on for editing target genes. This system was generally introducing *cas9* gene with plasmid vector and expressed in mycelium, and as a result the transformants becomes gene modified organisms. Considering the utilization in industry, we developed the genome editing method by directly Cas9 introducing into the experimental strain of RIB40 and applied to various industrial strains.

We chose 5-FOA selectable *pyrG* in *A. oryzae* for genome editing target. Cas9 and *pyrG* single guide RNA (sgRNA) are mixed and formed ribonucleoproteins (RNPs), then introduced into *A. oryzae* by conventional protoplast-PEG method. 289 candidates were selected on 5-FOA containing plates. We confirmed the sequence of *pyrG* locus in 24 candidates and found that about half of the strains occurred 1bp deletion at target locus, indicating that genome editing was succeeded by this method. We further examined the amount of Cas9 use, and found that more than 10 mg is enough for *A. oryzae* genome editing by this method.

To confirm the practicality of the method, we carried out *pyrG* genome editing against some *A. oryzae* industrial strains, such as OIS01 (current Japanese Sake brewing), RIB128 (ancient Sake brewing), RIB163 (Japanese Sake brewing) and RIB915 (Soy sauce making) strains. We acquired genome editing candidates in the all industrial strains, indicating that this Cas9 direct introducing genome editing method is useful for molecular breeding of *A. oryzae* industrial strains.

**369F Functional and subcellular localization analyses of transcription factors, AtrR and SrbA, involved in azole drug resistance in Aspergillus oryzae.** K. Sugiyama¹, A. Ohba-Tanaka¹, D. Hagiwara², S. Kawamoto², M. Tanaka¹, T. Shintani¹, K. Gomi¹ 1) Graduate School of Agricultural Science, Tohoku University, Sendai, JP; 2) Medical Mycology Research Center, Chiba University, Chiba, JP.

We previously demonstrated that a novel Zn(II)₂Cys₆ transcriptional factor, AoAtrR, regulates gene expression of the ABC transporters that would function as drug efflux pumps and contributes to the azole drug resistance in *Aspergillus oryzae*, and a disruption mutant of the *AoatrR* ortholog (*AfatrR*) in *Aspergillus fumigatus* was similarly hypersensitive to azole drugs. In addition, RNA-seq analysis in *A. fumigatus* showed that AtrR regulated several ergosterol biosynthetic genes including *erg11*. Surprisingly, most of these ergosterol biosynthetic genes regulated by AtrR were nearly identical to those regulated by the basic helix-loop-helix transcription factor, SrbA (1). Therefore, we are interested in and investigate the difference in function between AtrR and SrbA in *Aspergillus oryzae*.

The expression of ergosterol biosynthetic genes such as *erg11*, *erg24*, and *erg25*, as well as ABC transporter genes, was significantly down-regulated in the *AoatrR* disruptant. Similarly, *AosrbA* disruption resulted in remarkable down-regulation of ergosterol biosynthetic genes same as in *A. fumigatus*, but not ABC transporter genes. In contrast, *AoatrR* overexpression did not resulted in up-regulation of ergosterol biosynthetic genes. These results suggested that AtrR and SrbA coordinately regulate ergosterol biosynthetic genes in aspergilli. However, *AoatrR* or *AosrbA* disruption had apparently no effect on another gene expression level, suggesting their expression would be regulated independently of each other. On the other hand, the *AoatrR* disruptant was more hypersensitive to azole drugs compared to the *AosrbA* disruptant and sensitive comparable to the *AoatrR/AosrbA* double disruptant, suggesting that hypersensitivity of the *atrR* disruptant to azole drugs is attributed not only to lowered ergosterol levels owing to down-regulation of ergosterol biosynthetic genes, but also to reduced efflux transport of the drugs owing to down-regulation of ABC transporter genes. Subcellular localization analysis of AoAtrR and AoSrbA showed that AoAtrR was constitutively localized in the nucleus and AoSrbA was likely localized in the nuclear envelope and/or
endoplasmic reticulum.

(1) Hagiwara et al., *PLoS Pathogens*, in press.

**370W Relationship between stability and subcellular localization of carbon catabolite repression regulator, CreA, in Aspergillus oryzae.** M. Tanaka, T. Shintani, K. Gomi Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, JP.

Aspergillus oryzae can produce considerable amounts of amylolytic enzymes in the presence of maltose, but expression of amylolytic enzyme genes is repressed by carbon catabolite repression (CCR) in the presence of glucose. CCR in filamentous fungi is regulated by the C<sub>2</sub>H<sub>2</sub>-type transcription regulator, CreA. When the 3FLAG-fused CreA was expressed under the control of the *creA* own promoter in A. oryzae, abundance of 3FLAG-CreA protein was dramatically reduced after addition of maltose or xylose, whereas this reduction was not observed after glucose addition. To examine the stability of CreA protein, 3FLAG-CreA was expressed under the control of the thiamine-repressible *thiA* promoter, and half-life of 3FLAG-CreA protein after addition of various sugars was measured.
After addition of maltose, xylose, and fructose, half-life of 3FLAG-CreA protein was calculated as approximately 11 minute. On the other hand, half-life of 3FLAG-CreA protein after glucose addition was approximately 25 minute. These results indicated that 3FLAG-CreA was stabilized by glucose addition. To investigate the relationship between stability and subcellular localization of CreA, we generated the mutated CreA harboring mutation in nuclear export signal (NES). Fluorescence microscope analysis of GFP-fused CreA showed that this mutated CreA remained into the nucleus after maltose addition, although intact CreA was exported to the cytosol. After maltose addition, half-life of 3FLAG-CreA harboring mutation in NES was calculated as approximately 33 minute. These results suggested that CreA was rapidly degraded in the cytosol under the CCR non-inducing condition.

This study was supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry, and Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry.

**371T Intron within 5' untranslated region enhances transcription of the enolase-encoding gene (enoA) in Aspergillus oryzae.** Taishi Inoue, Hiroki Toji, Mitsuru Takama, Mizuki Tanaka, Takahiro Shintani, Katsuya Gomi Tohoku University, Sendai, Miyagi, Japan.

**Back ground:** The glycolytic pathway is the primary metabolic process which is essential to metabolize a wide range of carbon sources in fungi. Hence, it is important for fungal survival to optimize the level of glycolytic genes expression in response to various environments. Interestingly, in *Aspergillus oryzae*, several genes which are involved in both glycolysis and gluconeogenesis, have alternative transcription start sites (TSSs). In addition, we demonstrated that selection of TSSs was dependent on two types of carbon sources; one is metabolized via glycolysis such as glucose and fructose, and another via gluconeogenesis such as acetate and ethanol. Particularly, stringent selection of alternative TSS was observed in the enolase-encoding gene (*enoA*). The similar transcriptional control of enolase gene was suggested in *Aspergillus nidulans* (Hynes et al., 2007). These findings seem to be important because it provides novel insight into environmental adaptation in the stage of transcriptional regulation of glycolytic genes in *Aspergillus* spp. However, the molecular details of alternative TSS selection remain to be elucidated. Remarkably, in *enoA*, there is an intron containing a downstream TSS (dTSS) within 5' untranslated region (5' UTR) when it is transcribed from an upstream TSS (uTSS). The length of the intron is 440 bp and quite long in fungi. In this study, we investigated the importance of this 5' UTR intron in *enoA* expression.

**Results:** To this end, we analyzed the enoA promoter plus 5' UTR whose intron was deleted (*PenoA-Δi*) or mutated at a splice site (*PenoA-issm*) using GUS reporter system. Under culture condition with acetate where transcription from uTSS is induced while transcription from dTSS is suppressed, both the GUS activity and the mRNA level were significantly decreased in *PenoA-Δi*. On the other hand, in *PenoA-issm*, the mRNA level was unaffected but the GUS activity was almost lost, presumably caused by emergence of uORF within intron unspliced. These results indicated that the deletion of dTSS within intron does not contribute to the reduction of gene expression in *PenoA-Δi*. Additionally, when the *enoA* gene was expressed by *PenoA-Δi* under condition that a resident *enoA* expression is suppressed, a resulting strain showed a significant reduction of the mRNA transcribed from uTSS and a poor growth under acetate culture condition. These results suggested that 5' UTR intron enhances the *enoA* transcription from uTSS in *A. oryzae*.

**372F Light-induced gene expression in Botrytis cinerea involves GATA-transcription factors and the stress-activated MAP kinase module.** Julia Schumacher¹, Kim Cohrs¹, Adeline Simon², Jaafar Kilani², Muriel Viaud², Sabine Fillinger² 1) IBBP, WWU, Muenster, Germany; 2) BIOGER, INRA, Grignon, France.

Botrytis cinerea is a plant pathogen that exhibits prominent light responses including the formation of the reproduction structures (photomorphogenesis), secondary metabolites/pigments, and antioxidant enzymes. A complex regulatory network of photoreceptors, transcription factors (TFs) and chromatin modifiers is supposed to initiate, transmit, and fine-tune the responses to different wavelengths of light on the transcriptional level that finally leads to the observable phenotypes. As the formation of the reproduction structures is strictly regulated by light in this fungus - conidia are formed in the light, sclerotia in the dark - the output can be easily monitored. The GATA-type TFs BcWCL1 (as part of the White Collar complex (WCC)) and BcLTF1 are important regulators as their deletions result in light-independent conidiation ("always conidia") due to the deregulation of BcLTF2 (Schumacher et al. 2014; Canessa et al. 2013; Cohrs et al. 2016). Study of light-induced gene (LIG) expression in both deletion mutants highlighted the role of the TFs in activating EARLY and in repressing LATE LIG expression, including *bcltf2* encoding the master regulator of conidiation and further LTFs that may function downstream or in parallel with BcLTF2 in regulating the conidiation process. As the group of LIGs also contained genes that are induced by various stresses in a BcSAK1-dependent manner, the phosphorylation status of the stress-activated MAPK BcSAK1 was studied and shown to increase after exposure to light. Deletion of *bcsak1* impairs LIG expression suggesting that BcSAK1 functions as a co-activator of the WCC in inducing EARLY genes, and as a component releasing the LATE promoters (e.g. *bcltf2*) from repression by BcLTF1 and the WCC resulting in the expression of the conidiation genes. This preliminary model is in agreement with the observations that the deletion of BcSAK1 results in a "never conidia" phenotype (Segmüller et al. 2007), and that the exposure of the wild type to heat and osmotic stress bypasses the requirement of light for conidiation.
**373W Investigation of six conserved transcriptional factors in *Candida tropicalis* in regulation of hyphae formation and biofilm development.** Y. CHEN, C. Lin Biochemical Science and Technology, National Taiwan University, Taipei, TW.

*Candida* species are opportunistic fungi, which can cause superficial and systemic infections in immunocompromised individuals. It has been known that regulatory network composed of Bcr1, Brg1, Efg1, Ndt80, Rob1 and Tec1 is involved in biofilm development in *C. albicans*. In this study, we compared the function of the six orthologous genes in *C. tropicalis* to those of *C. albicans* in biofilm and hyphae formation. Results showed that deletion of *BRG1*, *EFG1* or *TEC1* gene resulted in biofilm development deficiency and reduced its hyphae formation significantly in both *C. tropicalis* and *C. albicans*. Although mutant strains of *bcr1Δ*, *ndt80Δ*, and *rob1Δ* of *C. tropicalis* also caused a significant reduction in the biofilm mass, the hyphal formation displayed distinct outcomes compared to the mutants strains of orthologous genes in *C. albicans*. We therefore used cross-species gene transfer strategy to investigate their roles in biofilms and hyphae between two *Candida* species. Transformation of *C. tropicalis* *BCR1*, *NDT80*, and *ROB1* into each corresponding mutant of *C. albicans*, respectively, showed that *CtROB1* is unable to recover biofilm formation and filamentation. Reintroduction of the functional *CtBCR1* into *Cabcr1Δ* did not affect hyphae formation, but restored the developmental process of biofilms. Under serum condition, 64.3% and 41.7% of the wild-type strains of *C. albicans* and *C. tropicalis* cells formed filaments, respectively. Interestingly, mutant strains of *Candt80Δ* and *Ctndt80Δ* caused biofilm defects but had 24.5% and 63.1% of filamented cells in *Candt80Δ* and *Ctndt80Δ*, respectively, suggesting that Ndt80 in *C. tropicalis* might be a negative regulator in filamenetation. Thus, the results imply that the Ndt80 has the conserved function in biofilm formation, but the entire hyphal regulatory circuit might be different between two *Candida* species. Indeed, our results further demonstrated that expression of *CaNDT80* into *Ctndt80Δ* could repress the filamentation ability of the *Ctndt80* mutants. Taken together, our study demonstrates that each single transcriptional gene holds the conserved function in biofilm and in filamentation, but have evolved a sophisticated regulatory network by a group of factors for the control of hyphae formation.

**374T ZCF32, a Zn(II)2Cys6 transcription factor, suppresses the virulence of *Candida albicans*.** P. Kakade¹, V. Nagaraja¹,², K. Sanyal² 1) Indian Institute of Science, Bangalore-560012, Karnataka, India; 2) Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore-560064, Karnataka, India.

*Candida albicans*, an opportunistic human pathogen, carries an expanded family of Zn(II)2Cys6 zinc binuclear cluster transcription factors. Zcf32, a Zn(II)2Cys6 transcription factor, is the member of this expanded family of proteins. In the present study, we intend to gain insights into the role(s) played by this transcription regulator in the virulence of *C. albicans*. Global transcriptome analysis carried out for the *zcf32/zcf32* null mutant and wild-type revealed that biofilm development is the most altered pathway followed by iron homeostasis and hyphal development pathways. To delineate the functional correlation between *ZCF32* and the different traits regulated by it, we determined the set of genes directly regulated by Zcf32. Our data suggest that Zcf32 regulates biofilm formation by repressing the expression of adhesins, chitinases and a significant number of other GPI-anchored proteins. We have established that there is lesser recruitment of Zcf32 on the promoters of biofilm genes in biofilm condition compared to the planktonic mode of growth. This shows that the transcription factor *ZCF32* negatively regulates biofilm development in *C. albicans*. Iron homeostasis maintenance is considered as an important fitness attribute in *Candida* pathogenesis. While growth studies showed that *zcf32/zcf32* null mutant grows as good as wild-type in low as well as high iron condition, the molecular data hints at the role of Zcf32 in high iron homeostasis maintenance. Lastly, the virulence regulation of the pathogen by this transcription factor was studied in mice model of candidiasis. Notably, *zcf32/zcf32* null mutant displayed hypervirulence in comparison to the wild-type. Thus, *C. albicans*, a gut commensal as well as a pathogen, has evolved with a negative regulator of virulence which might have helped the organism to maintain the commensal state.

**375F Functional Analysis of the Cph1-regulated Genes of *Candida albicans* White Cells during the Response to Pheromone.** F. Deng, C. Lin Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, Taipei, TW.

*Candida albicans* is the most important fungal pathogen in human, particular in some immunocompromised patients. The propensity for causing *C. albicans* infection has been closely linked with its ability to form biofilms. Central to understanding the *C. albicans* behavior is the white-opaque phenotypic switch, in which cells can undergo an epigenetic transition between the white state and the opaque state. The phenotypic switch regulates multiple properties including biofilm formation, virulence, and sexual mating. In particular, it is now apparent that sexual pheromones secreted from opaque cells can initiate biofilm formation in white cells. Our studies have identified the transcriptional regulator of pheromone-induced cell adhesion as Cph1 (Ste12). However, the mechanism of how white cells in response to pheromone remains obscure. Based on the array profiling, we have identified 10 downstream genes regulated by Cph1 in the *C. albicans* white state, but not in the opaque state. We therefore hypothesized that these genes might play important roles in pheromone-induced white response specifically. Deletion of each gene revealed that most of mutant strains affected the pheromone-stimulated cell adhesion in white cells. In particular, *orf19.1539Δ*, *orf19.1725Δ* and *orf19.3643Δ* showed a significant reduction in cell adhesion during the response to pheromone. Due to adherent ability is an important first step in establishing biofilm formation. *orf19.1539Δ*, *orf19.1725Δ* and *orf19.3643Δ* were further investigated to understand if each of them is also involved in the formation of conventional biofilms. Results showed that mutant strains of *orf19.1725Δ* lacked its ability to form biofilms and repressed hyphal formation. Introduction of a functional copy of *ORF19.1725* returned its function of biofilm development and hyphal formation. Together, our data has discovered some genes that might be important for the white cell response, but not for the opaque cell response. It might provide a clue that why white and opaque cells display distinct behaviors during the response to pheromone.

**376W DNA Methylation Remodeling in *Candida albicans* Plays a Key Role in Epigenetic Regulation of Phenotype- Dependent Pheromone Responses.** S. Hoo¹, Y. Huang², A. Archana², P. Chen², C. Lin¹ 1) Biochemical Science and Technology, National Taiwan University, Taipei, TW; 2) Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan.

*Candida albicans* is the most frequently isolated fungal pathogen in immunocompromised patients with hospital-acquired infections, as the phenotypic plasticity enables easy adaption of *C. albicans* to different niches in immunodeficient patients. Among the phenotypic plasticity, there is a heritable and reversible phenotypic switching between white and opaque cells, called white-opaque switching. Herein, mating competent opaque cells secrete sexual pheromone to promote opaque cell mating projection, the process is called opaque cell pheromone response. Besides, the pheromone can also stimulate cell adhesion and sexual biofilm formation of mating incompetent white cells through white cell pheromone response. Interestingly, white and opaque cells are sharing the same pheromone signaling pathway and major transcription factor Cph1 despite having different cell specific response and gene expression during pheromone stimulation.
According to the previous research, DNA methylation efficiently regulates expression of cell specific gene between different cell types of *C. albicans*. In our study, the results of AzaC (5-azacytidine, DNA methylation inhibitor) treatment show that DNA demethylation inhibits white cell pheromone response, which is associated with downregulated white cell pheromone response genes (*EAP1* and *PBR1*). By contrast, opaque cell pheromone response is raised by DNA demethylation and opaque cell pheromone response genes (*FUS1* and *FIG1*) are also upregulated. Also, whole genomic bisulfite sequencing and RNA sequencing data further suggest that DNA methylation remodeling (include critical DNA demethylation and *de novo* methylation process) regulates different gene expressions and causes distinct cell behaviors in white and opaque cells during pheromone stimulation. Therefore, we suppose that DNA methylation remodeling in *C. albicans* may play a key role in regulating phenotype-dependent pheromone responses.

**377T The role of *C. neoformans* chitin deacetylases in chitosan mediated virulence.** *R. Upadhya*¹, *W.C Lam*¹, *L.G Baker*¹, *M.J Donlin*², *C.A Specht*³, *J.K Lodge*¹ 1) Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO; 2) Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine; St. Louis, MO; 3) Department of Medicine, University of Massachusetts Medical School, Worcester, MA.

Chitosan is one of the major virulence factors of *C. neoformans*. Mutant strains deficient in chitosan are avirulent in mice and are rapidly cleared from the host. Enzymatic conversion of chitin to chitosan is catalysed by chitin deacetylases. We have discovered that deletion of three deacetylase genes: *CDA1*, *CDA2* and *CDA3* are required for generating a chitosan deficient *C. neoformans* strain. This *cda1?2?3?* strain is avirulent and when cleared induces protective immunity to a lethal infection by its virulent, progenitor strain KN99. To further understand the role of individual chitin deacetylases in fungal pathogenesis we generated mutant strains deleted of either a single CDA gene or combinations of two CDA genes. Individual CDA deletion strains did not show any defect in the amount of chitosan under standard growth conditions, in vitro. However, when tested using inhalation model of cryptococcosis, we found that only *cda1?* was avirulent and both *cda2?* and *cda3?* exhibited wild-type virulence. These results indicate that *C. neoformans* *CDA1* is the major chitin deacetylase that is critical for establishing infection and eventual mortality to infected animals. The importance of *CDA1* is further supported by its specific upregulation during growth in the infected lung while *CDA2* and *CDA3* exhibit downregulation. In addition, *cda1?* cells in the infected lung produced less chitosan than KN99. Interestingly, the infected host lung was not able to clear *cda1?* cells. Of the three double deletion combinations only *cda1?2?* was also cleared. These data support a minor role of *CDA2* in the persistence of fungus inside the host. In conclusion, in *C. neoformans* the chitosan produced by *CDA1* is critical for fungal virulence.

**378F Botrytis cinerea, more than just a pathogen: a new platform to dissect integration of environmental signals and circadian mechanisms.** *Paulo Canessa*¹,³, *Montserrat Hevia*²,³, *Hanna Müller-Esparza*²,³, *Luis Larrondo*²,³ 1) Centro de Biotecnologia Vegetal, Universidad Andres Bello, Santiago, RM, CL; 2) Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, RM, CL; 3) Millennium Nucleus for Fungal Integrative and Synthetic Biology (FISB), Santiago, RM, CL.

BcWCL1 and BcFRQ1 are two major components of the recently characterized *Botrytis cinerea* circadian clock. These molecular machineries have emerged at least three times during evolution and are cell-contained time-devices that allow temporal compartmentalization of several functions, both at the cellular level (i.e. gene expression) and at the systemic level (i.e. general physiology, metabolisms and defense mechanisms). As previously demonstrated, BcWCL1 serves light-dependent developmental-associated functions, while BcFRQ1 is the central circadian clock pacemaker that impacts time-dependent virulence in *B. cinerea*. An exhaustive bioinformatic examination of several fungal genomes has allowed the identification of the clock negative element (FRQ) outside a couple of previously identified fungal clades. In the case of BcFRQ1, close examination of the protein sequence reveals conservation of several domains and regions of low structural complexity, thus supporting its function as the clock pacemaker. Nevertheless, both phenotypical and molecular data, derived by comparison of genome-wide expression data of *bcwcl1* and *bcfrq1* null mutants, indicate new "supra-circadian" functions for this clock protein. Thus, in addition to its role in orchestrating daily changes in the *B. cinerea* virulence potential, BcFRQ1 appears to be at the crossroads between nitrogen metabolism and asexual/sexual developmental programs. FUNDING: MN-FISB NC120043, FONDECYT Inicio 11140678 and FONDECYT 1131030.

**379W Interconnected network of circadian rhythms and glycogen metabolism in *Neurospora crassa*.** M. Baek¹, S. Virgilio², A. Dovzhenok³, T. Lamb⁴, O. Ibarra⁴, S. Lim³, D. Bell-Pedersen⁴, M.C. Bertolini², *C.I. Hong*¹ 1) Department of Molecular & Cellular Physiology, University of Cincinnati, Cincinnati, OH, USA; 2) Departamento de Bioquímica e Tecnologia Química, Instituto de Química, Universidade Estadual Paulista, UNESP, 14.800-060, Araraquara, São Paulo, Brazil; 3) Department of Mathematical Sciences, University of Cincinnati, Cincinnati, OH 45221-0025, USA; 4) Department of Biology, Texas A&M University, College Station, TX 77843, USA.

Circadian rhythms provide temporal information to other cellular processes including metabolism, which align organisms' physiological processes with the external environment. In this report, we utilized mathematical modeling and experimental validations to investigate molecular mechanisms that link circadian rhythms and glycogen metabolism in *Neurospora crassa*. We discovered complex regulation of the expression of key enzymes that regulate glycogen metabolism, glycogen synthase (*gsn*) and glycogen phosphorylase (*gpn*), by three different transcription factors (CSP-1, VOS-1, and WCC). This combinatorial TF regulation of *gsn* and *gpn* results in robust circadian oscillations of glycogen abundance, which is abolished in circadian arrhythmic mutants (*frq*<sup>k0</sup> and *wc-1*<sup>k0</sup>). Our findings elucidate key players and mechanisms that regulate rhythmic abundance of glycogen, which is critical for glucose homeostasis.

**380T Analysis of circadian regulated transcripts under cellulose growth conditions in Neurospora crassa.** *Adrienne Mehalow*¹, Alexander Crowell¹, Kerrie Barry², Igor Grigoriev², Jennifer Loros¹, Jay Dunlap¹ 1) Molecular and Systems Biology, Dartmouth Medical School, Hanover, NH; 2) US Department of Energy Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA 94598.

Circadian clocks are highly conserved in eukaryotic organisms and confer the ability to anticipate regular changes in the environment resulting from the 24 hour day on earth. *Neurospora crassa* has been utilized to investigate the molecular clockworks for more than 30 years, due to readily observable circadian phenotypes, availability of mutants, and ease of manipulation in the lab. The *Neurospora* clock consists of a core clock oscillator with interconnected transcription and translation feedback loops. This oscillator produces a self-sustaining rhythm, is entrainable to environmental cues, and regulates the behavior of downstream clock-controlled genes (*ccgs*). *Neurospora* is capable of metabolizing a wide variety of carbon sources, although is most commonly cultured on glucose. RNA-seq