SRAP markers as an alternative tool for Alternaria classification.

Alternaria is one of the main fungal contaminants of cereal grains worldwide with the potential to produce mycotoxins hazardous to human and animal health. Many studies have been carried out to characterize Alternaria sp.-grp. using traditional morphology or polyphasic approach, but a good correlation between morphological sp.-grp., molecular, and chemotaxonomic groups has not always been achieved. For this reason, this study aimed to investigate the usefulness of a cheaper alternative tool, SRAP markers, in identifying Alternaria sp.-grps. obtained from Argentinean barley grains and to compare it with preliminary characterization using morphological traits, phylogeny, and metabolite profiles. Fifty-three Alternaria isolates from barley grains of the main producing regions of Argentina were analyzed with four combinations of SRAP markers. The UPGMA dendrogram, based on the Simple Matching similarity coefficient, revealed three distinct groups. SRAP markers allowed the separation of Alternaria from Infectoriae sections in agreement with the results of a polyphasic approach previously made. Besides, isolates of A. arborescens sp.-grp. were clustered in a separate group from isolates of A. tenuissima and A. alternata sp.-grp., which were grouped in the same cluster. SRAP markers are a recommended tool for classifying Alternaria isolates because of its simplicity, reliability, and cost-effectiveness compared to other molecular markers.

Interaction of methyl-jasmonate and Fusarium poae in bread wheat.

Fusarium Head Blight (FHB) is a devastating disease that affects the grain yield and quality of essential crops such as wheat. In the last years, some Fusarium species have acquired particular importance as Fusarium poae. However, studies to evaluate F. poae-wheat interaction are still scarce. The interaction between F. poae and two bread wheat cultivars with different resistance levels against FHB was evaluated. Moreover, the application of methyl-jasmonate (MeJA) was evaluated as a possible tool to reduce the fungal presence. Our results showed that the MeJA treatment is isolate-dependent, reducing F. poae fungal growth. A decrease in fungal biomass was observed in the susceptible cultivar after MeJA application; however, no differences between inoculated and inoculated-MeJA treatments were observed in the resistant cultivar. Finally, the F. poae inoculation induces the expression of PR1-1 and PDF 1.2, being early in the resistant cultivar compared to the susceptible ones. The application of MeJA combined with the F. poae inoculation increased PR1-1 and PDF1.2 expressions in resistant cultivars. To our knowledge, this is the first study that evaluates the interaction between F. poae and wheat and the MeJA treatment as a possible management strategy against this important pathogen.

Fusarium pseudograminearum Associated with Barley Kernels in Argentina.

Barley (Hordeum vulgare L.), one of the most widely grown winter cereal crops in Argentina, is primarily grown for use as malted barley for the beer industry. In December 2010, a survey of fungi was conducted in a barley (cv. Shakira) seed lot in a field located in Tres Arroyos, Buenos Aires, Argentina. A sample of 400 seeds was surface sterilized (70% EtOH for 2 min and 5% NaClO for 2 min), rinsed twice in sterilized distilled water, plated on potato dextrose agar (PDA), and incubated at 24 ± 2°C in a 12-h dark/light cycle. One isolate that was morphologically similar to Fusarium graminearum was observed after 6 days of incubation. The isolate was transferred onto PDA and carnation leaf agar (CLA) substrates and grown with the same conditions as described above. On PDA, the isolate produced abundant, white-to-yellow-to-red, aerial mycelium and formed red pigments in the medium. On CLA, macroconidia were abundant, relatively slender and almost straight to moderately curved, and commonly five to six septate. Microconidia were not observed. Chlamydospores were observed after 3 weeks. The fungus was initially identified as F. graminearum on the basis of morphology of the asexual stage (1). Pathogenicity was conducted using a hand sprayer to inoculate five barley (cv. Shakira) heads in potted plants with a 5-ml asexual spore suspension (1 × 104 conidia per ml). Two heads were sprayed with sterile distilled water as a control. Plants were covered with polyethylene bags and incubated for 3 days in a growth chamber under a 12-h day/dark cycle at 22 ± 2°C. Plants were unbagged and moved into a greenhouse. Noninoculated spikelets were asymptomatic and inoculated spikelets showed discoloration or a tan-to-dark brown necrosis. The fungus was reisolated from symptomatic kernels. DNA of the isolate was extracted (3) and the isolate was identified to species by sequencing the reductase (RED), trichothecene 3-O-acetyltransferase (tri101), and translation elongation factor (TEF) regions (4). The sequences were compared with those in GenBank. The RED sequence (Accession No. JQ350697) showed 100% similarity, the tri101 (Accession No. JQ350698) showed 99% similarity, and the TEF (Accession No. JQ350699) showed 100% similarity with several F. pseudograminearum sequences. Additionally, the isolate was tested for the potential to produce deoxinyvalenol (DON) using a PCR approach that allows identification of two acetylated forms of DON: 15-acetyl-DON (15-ADON) and 3-ADON (2). A PCR product indicative of a 3-ADON genotype was obtained. To our knowledge, this is the first report of F. pseudograminerum associated with barley kernels in Argentina. Considering its potential to cause head blight and product mycotoxins, a large-scale survey of F. pseudograminearum on barley crops in Argentina is underway. A voucher culture (No. 1154) has been deposited in the Culture Collection of the La Plata Spegazzini Institute. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, UK. 2006. (2) A. Quarta et al. Food Addit. Contam. 22:309, 2005. (3) S. A. Stenglein and P. A. Balatti. Physiol. Mol. Plant Pathol. 68:158, 2006. (4) T. J. Ward et al. Fungal Genet. Biol. 45:473, 2008.

Fusarium tricinctum Associated with Head Blight on Wheat in Argentina.

Wheat (Triticum aestivum L.), the most widely grown winter cereal crop in Argentina, is grown on 5 million ha. Fusarium species affect yield and grain quality because of mycotoxins. In December 2009, a screen of fungal species in wheat seeds from a field in Azul, Buenos Aires, Argentina was conducted. Four hundred seeds were surface sterilized by dipping successively into 70% ethanol for 2 min, 5% sodium hypochlorite for 2 min, and finally rinsing twice in fresh sterilized distilled water. The seeds were plated on potato dextrose agar (PDA), pH 6, and incubated at 24 ± 2°C with exposure to 12-h alternate cycles of darkness and light. Eight isolates morphologically similar to Fusarium species were observed after 6 days of incubation. For identification, monosporic isolates were transferred onto PDA and carnation leaf agar (CLA) to grow at the conditions described above (1). One isolate, when grown on PDA, rapidly produced abundant, dense, white, aerial mycelium that became pink with age and formed red pigments in the medium. On CLA, macroconidia were abundant, relatively slender, curved to lunate, and three to five septate. Microconidia were abundant, napiform, oval or pyriform, zero to one septate, and commonly clustered in false heads. Chlamydospores were absent. The fungus was identified as Fusarium tricinctum (Corda) Saccardo on the basis of fungal morphology (1). To complete Koch's postulates, the pathogenicity of the fungus was tested by spraying five healthy inflorescences (on average 16 spikelets per spike) of wheat with a 5-ml suspension (2 × 105 conidia per ml). Another two healthy inflorescences were sprayed with sterile distilled water. Plants were placed in a growth chamber with a 12-h photoperiod at 22 ± 2°C, covered with polyethylene bags that were removed after 3 days, and then moved to a glasshouse. The same procedure was repeated. While control inflorescences were asymptomatic, inoculated inflorescences showed a mean of five bleached spikelets per spike. By using the methodology described above, the fungus was reisolated from all infected grains of inoculated plants but not from the controls. To confirm the morphological diagnosis, the genomic DNA of the isolate was extracted (3) and the internal transcribed spacer (ITS) and the translation elongation factor (TEF) regions were PCR-amplified using primer pairs ITS3/ITS4 (4) and EF-1/EF-2 (2), respectively. The sequences were compared with those in GenBank. The ITS sequence (Accession No. HM635739) showed 100% similarity with several F. tricinctum sequences (e.g., Accession Nos. HM068317, FN598932, and EF589873) but also with other Fusarium species such as F. acuminatum. The TEF sequence (Accession No. HQ214681) showed 99 to 100% similarity with Accession Nos. HM068307, EU744838, and EU744837 of F. tricinctum. To our knowledge, this is the first report of F. tricinctum on wheat in Argentina. This species is known to produce fusarin C, enniatins, and moniliformin toxins. Since F. tricinctum can infect different cereal grains, a large-scale survey of cereals from fields throughout Argentina is in progress. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, UK. 2006. (2) K. O'Donell et al. Proc. Nat. Acad. Sci. USA 95:2044, 1998. (3) S. A. Stenglein and P. A. Balatti. Physiol. Mol. Plant Pathol. 68:158, 2006. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.