Volatile organic compounds production by trichothecene non-producing Fusarium graminearum mutants

JUAN R. GIROTTI; GLADYS A. LORI; ISMAEL MALBRÁN; BARBARA SCHERM; CECILIA A. MOURELOS; QUIRICO MIGHELI

Florianópolis

Congreso; 5th International Symposium on Fusarium Head Blight - 2nd International Workshop on Wheat Blast; 2016

Fusarium graminearum Schwabe, the main fungal pathogen associated with Fusarium head blight (FHB), contaminates agricultural crops and commodities with mycotoxins, mostly the trichothecenes deoxynivalenol (DON), nivalenol (NIV), and their acetyl-derivatives. It has been demonstrated that DON plays a role in pathogenesis. The first cyclic intermediate in the biosynthetic pathway of trichothecenes is the trichodiene, which synthesis is catalyzed by the enzyme trichodiene synthase, codified by the TRI5 gene. During trichothecene metabolism several volatile organic compounds (VOC) are produced, which can be detected using the solid phase microextraction (SPME) technique coupled to capillary gas chromatography (CGC) mass selective detection (MS). With the objective of comparing the VOC production profiles of trichothecene producing and non-producing strains of F. graminearum, trichothecene nonproducing mutants were obtained using a split marker recombination approach and VOC were measured and identified using SPME-CGC-MS. In a DON producing F. graminearum strain, TRI5was replaced with a marker gene (hph, which confers resistance to hygromicin B) by protoplast transformation with two constructs each containing a portion of hph and of a ~500 bp region flanking either the upstream or downstream side of the target gene. Replacement of the gene was confirmed by PCR and Real-time qRT-PCR. Furthermore, decreased aggressiveness of the knock-out mutants was verified in a field test on point inoculated wheat spikes which were later checked for DON content using a commercial enzyme-linked immunosorbent assay (ELISA) kit. For VOC analysis, mutants were cultivated on 50 gr of 80% humidity (w/w) rice in 500-ml flasks. VOCs were extracted from the head space of fungal cultures 7 days after inoculation using a polidimethylsiloxane/divinylbenzene (PDMS/DVB) fiber for 30min. CGC-MS analysis was performed using a Hewlett Packard 6890 gas chromatograph coupled to an HP 5975C VL Agilent mass selective detector employing a non-polar HP-5 capillary column. Seventeen F. graminearum transformants were obtained of which 76% were confirmed to be (TRI5 mutants by PCR, qRT-PCR, aggressiveness tests (F = 23.28) and DON production (no detectable amounts of DON were observed). The remaining 24% of the transformants were ectopic mutants that behaved as the wild type strain in all tests. Both the wild type and ectopic strains showed the presence of several VOC in the elution zone of sesquiterpenes. A major peak eluting at ~ 15 min was identified as trichodiene by interpretation of its mass spectral fragmentation. On (TRI5mutants, on the other hand, neither trichodiene nor other sesquiterpene compounds were detected. The results obtained showed that TRI5 was correctly and efficiently replaced by the marker gene in the F. graminearum strain tested and that this replacement hindered the production of TRI and other sesquiterpenes by the mutants obtained.