Presence of Fusarium graminearum sensu stricto associated with triticale ( × Triticosecale Wittmack) in Argentina

Aim of study: To report the occurrence of Fusarium graminearum sensu stricto (s.s.) on triticale grains from field samples in Argentina and the potential mycotoxin production for these isolates. of study : Buenos Aires province, Argentina. Material and methods : A total of 40 samples from different crops (barley, rye, triticale, and wheat) showing Fusarium head blight symptoms were taken during 2017/2018 harvest season. Colonies with colour and mycelium similar to Fusarium were taken and were morphologically and molecularly identified. The potential to produce deoxynivalenol, 15-acetyl deoxynivalenol and zearalenones was determined. Also, the Koch’s postulates were used to evaluate the pathogenic capacity of the F. graminearum s.s . isolates in triticale. Main results : Two Fusarium isolates were identified morphologically as F. graminearum , which were confirmed molecularly by PCR using the specific Fg16 F/R primers pair and by sequencing red and tri101 genes. The sequences obtained were compared with those available in the NCBI database using BLAST tools, showing 99-100% homology with those belonging to F. graminearum s.s. The results demonstrated that F. graminearum s.s. isolates were pathogenic when triticale spikes were inoculated by spraying under greenhouse conditions. of s.s .


Introduction
Triticale (× Triticosecale Wittmack), developed years ago by plant breeders, is a cross between wheat (Triticum sp. L.) and rye [Secale cereale L. (M. Bieb)], that combines characteristics of their parental crops. For instance, as regard to bread-making quality, the amount of protein and gluten strongly correlates with strength and elasticity of the dough in wheat. However, in triticale, this correlation is lower because part of the protein comes from rye lacking the capacity to form gluten (Skovmand et al., 1984). Considering nutritional qualities, the main 2 in the plants producing unfilled grains and plant death (Desmond et al., 2006).
The presence of Fusarium spp. leads to mycotoxin production whenever the climatic conditions are optimal to allow that the pathogen produces these secondary metabolites. These mycotoxins have a different impact on consumers depending on the type of mycotoxin produced. Trichothecenes are the most important group of Fusarium mycotoxins strongly associated with chronic and fatal toxicoses of humans and animals being deoxynivalenol (DON) and nivalenol (NIV) the most prevalent in different substrates (Dejardins & Proctor, 2007). Zearalenones (ZEA) are other mycotoxins structurally different to trichothecenes and acutely less toxic, which have not been associated with any fatal mycotoxicoses. Each Fusarium species has the genetic potential to produce different mycotoxins, whose production will depend on the environmental conditions present during the pathogen development. A correct morphological and molecular characterization is needed to identify the causal agent of the disease and thus know what mycotoxin could be present if the temperature and humidity conditions are optimal for mycotoxin production. Therefore, the objective of this study was to report the occurrence of F. graminearum s.s. on triticale grains from field samples in Argentina and the potential mycotoxin production for these isolates.

Field description and fungal identification
Different crops including barley (Hordeum vulgare L.), rye, triticale, and wheat were sampled from different fields in 14 locations of Buenos Aires province, Argentina, during the 2017/2018 harvest season. A triticale sample with around 20% of visual FHB symptoms in its glumes was manually collected at the sampling point located at 37°2'7.08'' S, 62° 14'2.76'' W ( Fig. 1). The symptomatic grains were surface-disinfected by using 1% sodium hypochlorite solution, 70% ethanol and rinsed twice in sterile distilled water. Finally, grains were introduced on Petri dishes (90 mm) containing 2% potato dextrose agar (Laboratorios Britania S.A, Argentina) and incubated at 25 ± 2 °C in 12 h dark/light conditions for six days. After this incubation time, two colonies with colour and mycelium similar to Fusarium were taken and monosporic cultures were performed on Spezieller Nährstoffarmer Agar. Finally, these single spore isolates were transferred to Petri dishes containing carnation leaf agar as recommended for morphological Fusarium identification in the same conditions described above (Leslie & Summerell, 2006). A slide of spores was prepared and stained with Lactophenol Blue. The triticale grain component is starch with similar content to its parent, but with a better balance composition of amino acids showing advantages over the remaining crops (Villegas et al., 1980). Regarding physical characteristics and chemical composition of grains such as protein content, crude fiber, ether extract, free sugars, and ash, triticale is in general intermediate between wheat and rye (Peña, 2004). All these characteristics allow that triticale not only is used as animal feed but also for human food in the preparation of baking products, such as different types of bread, noodles, and softwheat type products (Oliete et al., 2010).
The capacity of producing a high amount of biomass and grain yield and the ability to adapt to different soils and climatic conditions allow the expansion of triticale to different regions. Hence, this crop is sown in different countries. Poland is the major producer with a production of 5.31 million tons (MT) followed by Germany (2.32 MT), Belarus (1.61 MT), France (1.59 MT) and China (0.98 MT) (http://www.fao.org/faostat/ es/?#data/QC). In Argentina, 499,300 ha were destined to triticale during the 2017/2018 harvest season with a production of 15,205 kg for human consumption and the remaining used for animal feed (http://datosestimaciones.magyp.gob.ar/). Last years, the global triticale production increased due to be a good source of protein and energy used mainly for animal feed.
Over a long time, triticale was considered more resistant to diseases than wheat and rye. Due to the expansion of the area sown and the cultivation time, the same pathogens present in its parents spread in this crop losing this acquired advantage (Arseniuk, 1996). Several diseases caused mainly by species belonging to Alternaria, Bipolaris, Dreschlera, and Fusarium genera have been reported in triticale (Arseniuk, 1996). When environmental conditions such as temperature and humidity are optimal for its occurrence, Fusarium head blight (FHB) is the prevalent disease in crops, which leads mainly to a reduction in yield and quality of grains. The most common pathogen able to cause this disease is F. graminearum s.s., whose presence has been reported in wheat, barley, and rye, among others. This species, together with at least other 15 species, is a member of the Fusarium graminearum species complex (FGSC), with F. graminearum s.s. being the most common species found in grasses (Aoki et al., 2012). Outside of this complex, F. avenaceum, F. culmorum, and F. poae have been frequently isolated from plants with FHB symptoms (Reis & Carmona, 2002). Other species known as F. pseudograminearum is mainly responsible for Fusarium crown rot, a prevalent disease where dry climatic conditions and conservation-farming practices are prevalent. The presence of this pathogen may interfere with the water and nutrient transports 3 Short communication: Fusarium graminearum s.s. associated with triticale in Argentina were purified by using PureLink PCR purification kit (Invitrogen, Leohne, Germany) and were sequenced using Big Dye Termination version 3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems, CA, USA) in an Applied Biosystems Sequencer (ABI/Hitachi Genetic Analyzer 3130).

Potential mycotoxin production
PCR reactions were carried out using primers previously described for NIV and DON genotypes. The NIV, DON, and the two 3-acetyl deoxynivalenol (3-ADON) and 15-acetyl deoynivalenol (15-ADON) genotypes were differentiated by using a multiplex PCR reaction with primers targeting portions of the tri7 and tri3 genes for NIV and DON genotypes, respectively with the same PCR conditions as described by Quarta et al. (2006). Primers based on tri13 gene designed by Chandler et al. (2003) were equally tested. Finally, PKS4 primers developed by target the PKS gene needed to ZEA production were used according to Meng et al. (2010). DNA of a F. graminearum s.s. isolate (3.6) characterized as 15-ADON and ZEA genotypes, one 3-ADON F. pseudograminearum (14/11) and one NIV F. meridionale isolate (NRRL 28436) were used as controls for PCR reactions (Castañares et al., 2016). NRRL isolates were kindly provided by the ARS Culture Collection. Products were examined by electrophoresis in 1.5% (w/v) agarose gels containing Gel-RedTM (Biotium, Hayward, CA, USA) at 80 V in 19 Trisborate-EDTA buffer at room temperature. Fragments were visualized under UV light. The size of the DNA fragments was estimated by comparing the DNA bands with a 100-bp DNA ladder (Genbiotech S.R.L.). fungal structures were observed and measured using an ocular micrometer disk in an optical microscope (Olympus CX31) at 40 magnification.

Molecular identification
Genomic DNA from two previously identified isolates (T1 and T2) was extracted based on the CTAB method (Stenglein & Balatti, 2006). These DNAs were used as templates in a F. graminearum-specific PCR using primers Fg16F and Fg16R with an annealing temperature of 62 °C according to Nicholson et al. (1998). Besides, PCR reactions were carried out using primers targeting portions of the reductase (red) and trichothecene 3-Oacetyltransferase (tri101) genes to identify the isolates to F. graminearum species level within the FGSC (O'Donnell et al., 2000). The primer sequence for RED primers were: REDF (5' AGA CTC ATT CCA GCC AAG 3') and REDR (5' TCG TGT TGA AGA GTT TGG 3'), while for TRI101 were: TRI101F (5' CAA GAT ACA GCT CG CAC C 3') and TRI101R (5' CTG GGT AGT TGT TCG AGA 3') with an annealing temperature of 57°C and 62°C, respectively. All PCR reactions were performed using 10-20 ng of DNA in a total volume of 25 µL containing 10× reaction buffer, 0.5 µM of the respective primers, 200 µM of each dNTP (Inbio-Highway, Tandil, Argentina), 2.5 mm MgCl 2 and 1.25 U of Taq DNA polymerase (Inbio-Highway, Tandil, Argentina). PCR conditions were 95 °C for 2 min for an initial denaturalization, followed by 30 cycles consisting of 94 °C for 30 s, 30 s at temperature previously described for each primer pair, 72 °C for 5 min and finally an elongation step at 72 °C for 10 min performing in an XP Thermal cycler (Bioer Technology Co.). PCR products toms were registered at 21 dpi. After physiological maturity, spikes from each treatment were threshed and cleaned manually. Symptomatic grains were surfacedisinfected and the fungus was re-isolated and identified morphologically as F. graminearum as described above.

Results and discussion
Based on morphological characteristics, the isolates obtained from triticale were identified as F. graminearum (Schw.). The presence of sporodochia formed by macroconidia with 5-6 septa with a width average value of 4.5-5 µm and a length average value of 50-60 µm was visualized. Moreover, abundant chlamydospore presence were visualized, which are frequently found in the macroconidia, while microconidia were not found (Leslie & Summerell, 2006). The results of F. graminearum specific PCR amplified a fragment of 400-500 pb as expected according to Nicholson et al. (1998). To confirm molecularly our results, the final sequences of red and tri101 fragments of T1 and T2 isolates were compared by Gel images were photographed with a digital DOC 6490 system (Biodynamics S.R.L., Buenos Aires, Argentina).

Pathogenicity test
The pathogenic capacity of two isolates (T1 and T2) was tested by Koch's postulates. For this purpose, the inoculum was produced in a liquid medium containing carboxymethyl cellulose with uniform agitation (100 rpm, 25 ± 2 °C and darkness), while triticale plants were grown in a greenhouse in 5 L pots (one plant per pot). During flowering stage, 5 mL of spore suspension (10 5 conidia/mL) were sprayed in the spike until run off, while control pots were sprayed with sterile distilled water ( Fig. 2A). The experiment design (inoculated and control treatment) was completely randomized with three replicates. Plants were covered with polyethylene bags for 96 h to ensure >85% of relative humidity and incubated in a greenhouse at 25 ± 2 °C. Spikes were photographed at 0, 2, 7, 14, 21 days post-inoculation (dpi) and at physiological maturity (Fig. 2). Finally, FHB symp-   2B). At 4 dpi, spikelets were completely affected ( Fig. 2C), being the spike totally affected at 14 dpi ( Fig 2E). At physiological maturity, grains were severely affected showing mycelial growth and unfilled grains (Fig. 2F). Therefore, this is the first report that shows the presence of F. graminearum s.s. in triticale in Argentina. Although several works have been developed using different crops inoculated with F. graminearum (barley, oat, rye, triticale, and wheat), scarce information are available about the natural occurrence of F. graminearum s.s. in triticale (Logrieco et al., 1990;Langevin et al., 2004;Ferreira Geraldo et al., 2006). Moreover, our results showed that both isolates amplified the 708 bp and 525 bp fragment corresponding to 15-ADON and DON genotypes, respectively, while no amplification was observed for 3-ADON and NIV genotypes (Fig.  3A). Confirming these results, the 282 bp fragment was also visualized by using the primers based on the tri13 genes, while no products were observed for NIV genotypes (Fig. 3B). As regard to ZEA production, T1 and T2 showed a 280 bp fragment associated with the potential to produce this mycotoxin (Fig. 3C). The potential capacity of producing mycotoxins such as 15-ADON and ZEA could have a severe impact on consumer health. DON has been reported to be protein synthesis inhibitors targeting dividing cells such as leukocytes causing altered response immunity on consumers, while ZEA is responsible of pathological changes in the reproductive system (Chung et al., 2003;Böhm et al., 2010). In southern Brazil, the production of mycotoxins by F. graminearum in small cereals was evaluated by Ferreira Geraldo et al. (2006). In this study, seven isolates were isolated from triticale seeds and the DON and ZEA production in vitro was detected. The response of winter and spring triticale genotypes was evaluated against the natural source of F. graminearum inoculum at five sites across Canada (Veitch