Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSSecretome analysis of Trichoderma harzianum cultivated in the presence of Fusarium solani cell wall or glucose. Marcelo HS Ramada 1,3 , Andrei SSteindorff 1 , Carlos Bloch Jr. 3 , Cirano J Ulhoa 2 . 1) Brasilia University, Cell Biology Department, Brasilia, DF, Brazil; 2) Federal University of Goias, BiochemistryDepartment, Goiania, GO, Brazil; 3) EMBRAPA CENARGEN, Mass Spectrometry Laboratory, Brasilia, DF, Brazil.Trichoderma harzianum is a fungus well known for its potential as a biocontrol agent of many fungal phytopathogens. The aim of this study was toevaluate the potential of T. harzianum ALL42 to control Fusarium solani, a phytopathogen fungus that causes several losses in common bean and soy cropsin Brazil and to evaluate the secreted proteins of T. harzianum ALL42 when its spores were inoculated and incubated in culture media supplemented (TLE)or not (MM) with nitrogen sources and in the presence or not of F. solani cell walls (FsCW). In the absence of FsCW, the media were supplemented withglucose (GLU). T. harzianum was able to control the phytopathogen growth and started to sporulate in its area after 7 days in a dual culture assay,indicating that it had successfully parasitized the host. T. harzianum was able to grow in TLE+FsCW, MM+FsCW, TLE+GLU, but unable to grow in MM+GLU.Protein quantification showed that TLE+FsCW and MM+FsCW had 45 and 30 fold, respectively, more proteins than TLE+GLU, and this difference wasobserved in the bidimensional gels, as the two supernatants from media supplemented with FsCW had around 200 spots and the one supplemented withglucose only had 18. TLE+FsCW and MM+FsCW had above 80% of spot similarity. A total of 100 proteins were excised from all three conditions andsubmitted to mass spectrometry analysis. 85 out of 100 proteins were identified. The only protein observed in all three conditions is a small protein, calledepl1, involved in eliciting plant-response against phytopathogens. An aspartic protease, previously described as related to mycoparasitism, was only foundwhen T. harzianum was grown with glucose. Gene expression was evaluated and confirmed the gel results. In the media supplemented with FsCW,different hydrolases like chitinases, beta-1,3-glucanases, glucoamylases, alpha-1,3-glucanases, and proteases were identified. Some proteins like a smallcystein-rich, alpha-L-arabinofuranosidase and NPP1, with no known function in mycoparasitism were also identified. T. harzianum ALL42 is able to inhibitthe growth and parasitize F. solani and showed a complex and diverse arsenal of proteins that are secreted in response to the presence of the cell walls,with novel proteins not previously described in mycoparasitism studies.Metabolic adaptation of the oomycete Phytophthora infestans during colonization of plants and tubers. Carol E. Davis, Howard S. Judelson. PlantPathology and Microbiology, University of California, Riverside, CA 92521.Phytophthora infestans is the causative agent of late blight and was responsible for the Irish famine in the 1840’s. Today it still continues to be a globalproblem and in the USA it has been reported that the economic loss on potato crops alone exceeds $6 billion per year. A successful phytopathogenicrelationship depends on the ability of the organism to adapt its metabolism during infection on various nutritional substrates (e.g., plant versus tuber) andat different times throughout infection when nutrients may be limiting. Investigation of this metabolic adaptation is key to understanding how P. infestanssucceeds as a pathogen. To do this, tomato plants and potato tubers were infected with zoospores using a “dipping” method. RNA was extracted at 3 dpiand 6 dpi and subsequently used in library preparation. Following this, the libraries were quality checked by analysis on a Bioanalyzer using a highsensitivity DNA chip. Using Illumina technology (50 bp, paired-end reads) RNA Sequencing was performed. For each sample an average of 262 million readswas obtained. As a reference for the in planta data, RNASeq was also performed on defined and complex media. Mining of the data shows that theexpression profiles of some pathways change, such as glycolysis and gluconeogenesis. Learning how metabolic adaptation occurs will prove useful in thedevelopment of novel control strategies for this plant pathogen.The fungi strike back: multidrug resistance in Aspergillus fumigatus and agricultural use of fungicides. Paul E. Verweij. Medical Microbiology, UMC StRadboud, Nijmegen, Netherlands.Aspergillus fumigatus is a saprophytic mould that causes a range of diseases in humans. The spectrum of diseases includes allergic conditions,aspergilloma and acute and chronic invasive aspergillosis. Acute invasive aspergillosis occurs typically in patients with compromised host defenses such asthose receiving treatment for leukemia. The management of invasive aspergillosis is very difficult as the diagnostic tools often lack sensitivity and thenumber of antifungal agents effective against the infection is limited. The azoles are the most important class of agents used for the treatment andprevention of invasive aspergillosis. Since 1998 resistance to medical triazoles has emerged in the Netherlands in clinical A. fumigatus isolates. Theseisolates commonly showed a multi-azole resistant phenotype and patients with azole-resistant aspergillosis failed to respond to azole therapy. Themortality rate of azole-resistant invasive aspergillosis was 88%. In more than 90% of resistant isolates a combination of changes in the target gene Cyp51Awas found: a substitution at codon 98 and a 34 bp tandem repeat in the promoter region (TR34/L98H). As person-to-person transmission is highly unlikelyin invasive aspergillosis, the dominance of a single resistance mechanism could not be explained by resistance development in azole-treated patients.Surveys in the environment showed that A. fumigatus isolates resistant to medical triazoles could be recovered from the environment, especially fromcultivated soil. The resistance mechanisms in these isolates were identical to those found in clinical isolates. Molecule alignment studies identified 5triazole fungicides that showed highly similar molecule structures to the medical triazoles. These 5 fungicides have been authorized between 1990 and1996, thus preceding the isolation of the first resistant clinical A. fumigatus isolate. TR34/L98H is increasingly reported in European countries, India, Chinaand Iran. Recently a new azole-resistance mechanism was found in Dutch patients and in the environment. The resistance mechanism again consisted of acombination of changes in the Cyp51A-gene: TR46/Y121F/T289A. This new resistance mechanism has spread in addition to TR34/L98H in the Netherlands,and has also been reported in the neighboring country Belgium. The selection of multiple azole resistance mechanisms in the environment posses a threatfor the use of azole drugs in the management of Aspergillus diseases in humans. Research that investigates the selection of azole resistance in A. fumigatusin the environment is urgently warranted.82