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.

First record of Fusarium verticillioides as an entomopathogenic fungus of grasshoppers.

Fusarium verticillioides (Saccardo) Nirenberg (Ascomycota: Hypocreales) is the most common fungus reported on infected corn kernels and vegetative tissues, but has not yet been documented as being entomopathogenic for grasshoppers. Grasshoppers and locusts represent a large group of insects that cause economic damage to forage and crops. Tropidacris collaris (Stoll) (Orthoptera: Acridoidea: Romaleidae) is a large and voracious grasshopper that in recent years has become an increasingly recurrent and widespread pest in progressively more greatly extended areas of some of in Argentina's northern provinces, with chemical insecticides being currently the only means of control. During February and March of 2008-09, nymphs and adults of T. collaris were collected with sweep nets in dense woodland vegetation at a site near Tres Estacas in western Chaco Province, Argentina, and kept in screened cages. F. verticillioides was isolated from insects that died within 10 days and was cultured in PGA medium. Pathogenicity tests were conducted and positive results recorded. Using traditional and molecular-biological methods, an isolate of F. verticillioides was obtained from T. collaris, and its pathogenecity in the laboratory was shown against another harmful grasshopper, Ronderosia bergi (Stål) (Acridoidea: Acrididae: Melanoplinae). The mortality caused by F. verticillioides on R. bergi reached 58 ± 6.53% by 10 days after inoculation. This is the first record of natural infection caused by F. verticillioides in grasshoppers.

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.

Fusarium proliferatum, a New Pathogen Causing Head Blight on Oat in Argentina.

Oat (Avena sativa L.) is widely grown (~200,000 ha) for livestock feed in Argentina. Fusarium spp. affect yield and commercial quality and can cause indirect losses because some Fusarium spp. produce mycotoxins. In December 2008, a study of oat seeds (cv. Graciela INTA) from Trenque Lauquen, Buenos Aires, Argentina was conducted. Seeds (400) were surface sterilized by dipping successively into 70% ethanol for 2 min, 5% sodium hypochlorite for 2 min, rinsed twice in fresh sterilized distilled water, plated on 2% potato dextrose agar (PDA) pH 6, and incubated at 24 ± 2°C with 12-h photoperiods. Six isolates morphologically similar to Fusarium spp. were observed after 6 days of incubation. For identification, monosporic isolates were transferred onto 2% PDA and carnation leaf agar (CLA) to grow with the conditions described above. Two isolates produced abundant, white, aerial mycelium and violet-to-dark (with age) pigments in the PDA. On CLA, macroconidia were abundant, slender, almost straight, thin walled, and usually three to five septate. Microconidia were abundant, usually single celled, oval or club-shaped in chains (less commonly in false heads) on monophialides and polyphialides. Chlamydospores were absent. The fungus was identified as Fusarium proliferatum (Matsushima) Nirenberg on the basis of fungal morphology (1). To complete Koch's postulates, the pathogenicity of the fungus was tested by spraying five healthy inflorescences of oat (cv. Graciela INTA) with a 5-ml suspension (2 × 105 conidia/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 and covered with polyethylene bags that were removed after 3 days and plants were moved to a glasshouse. This procedure was repeated. While control inflorescences were asymptomatic, inoculated inflorescences showed bleaching glumes that sometimes became necrotic with some grains that presented pale brown discoloration and necrotic areas. The fungus was reisolated from glumes and grains of inoculated plants and not from controls using the methodology described above. To confirm the morphological diagnosis, the genomic DNA of the isolates was extracted (3) and a PCR reaction with specific primers 5'-CTTTCCGCCAAGTTTCTTC-3'-forward and 5'-TGTCAGTAACTCGACGTTGTTG-3'-reverse was chosen (2) using the following cycling protocol: initial denaturation step at 95°C for 2 min; 30 cycles at 95°C for 30 s, 55°C for 30 s, 72°C for 45 s; final extension at 72°C for 2 min. Successful amplifications were confirmed by gel electrophoresis. Size of the DNA fragment was estimated using a 100-bp DNA ladder. The reaction was repeated three times. The expected size product (585 bp) was obtained, confirming the identification (2). To our knowledge, this is the first report of F. proliferatum on oat in Argentina. This species is known to produce fumonisins, beauvericin, fusaric acid, fusarins, and moniliformin toxins, among others. Since F. proliferatum can infect different cereal grains, a large-scale survey in the same and different fields is in progress. A voucher culture has been deposited in the LPSC (Culture Collection of the La Plata Spegazzini Institute) No. 1058. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, UK. 2006. (2) G. Mule et al. Eur. J. Plant Pathol. 110:495, 2004. (3) S. A. Stenglein and P. A. Balatti, Physiol. Mol. Plant Pathol. 68:158, 2006.