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.

Mixed cropping regimes promote the soil fungal community under zero tillage.

Fungi of yield soils represent a significant portion of the microbial biomass and reflect sensitivity to changes in the ecosystem. Our hypothesis was that crops included in cropping regimes under the zero tillage system modify the structure of the soil fungi community. Conventional and molecular techniques provide complementary information for the analysis of diversity of fungal species and successful information to accept our hypothesis. The composition of the fungal community varied according to different crops included in the cropping regimes. However, we detected other factors as sources of variation among them, season and sampling depth. The mixed cropping regimes including perennial pastures and one crop per year promote fungal diversity and species with potential benefit to soil and crop. The winter season and 0-5 cm depth gave the largest evenness and fungal diversity. Trichoderma aureoviride and Rhizopus stolonifer could be used for monitoring changes in soil under zero tillage.

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.

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.

Phylogenetic relationships of Fusarium poae based on EF-1 alpha and mtSSU sequences.

A molecular phylogenetic analysis of Fusarium poae isolates from South America (Argentina) and Europe (mainly England, Germany, Italy) was performed using 98 F. poae, four Fusarium culmorum, two Fusarium sporotrichioides and one Fusarium langsethiae isolates. Phylogenetic analyses were performed using nuclear (translation elongation factor 1-alpha, EF-1 alpha) and mitochondrial (mitochondrial small subunit rDNA, mtSSU) sequences. Partitioned (each dataset separately) and combined (EF-1 alpha+mtSSU) analyses did not reveal any clear correlations from the inferred branching topology, between the distribution of observed haplotypes and the geographic origin and/or host species. Results from the present study confirmed that isolates from F. poae form a monophyletic group, and the low variability within isolates from a broad geographic range suggests a common lineage history. Among F. poae isolates from Argentina, however, some were found to possess an insert within mtSSU with structural similarities to group IC2 introns. F. poae isolates differing by the presence/absence of a mtSSU insertion were characterized further by analysis of a portion of the Tri5 gene, but this sequence was unable to reveal variability. The presence of this insert only within isolates from Argentina suggests that evolutionary events (insertions/deletions) are probably taking place within the Argentinian F. poae isolates, and that the acquisition of this insert occurred after geographic isolation of the Argentinian and European populations.

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.

First Report of Angular Leaf Spot Caused by Phaeoisariopsis griseola on Phaseolus coccineus in Argentina.

Angular leaf spot (ALS), caused by Phaeoisariopsis griseola (Sacc.) Ferraris, is one of the most destructive and widespread problems of common bean (Phaseolus vulgaris L.) in Tucumán and other northwestern provinces of Argentina (4). Symptoms similar to those of ALS were observed during April 2005 on most plants of runner bean (P. coccineus L.) in an 80-ha field in Tafí del Valle, Tucumán (2,000 m above sea level). Leaf lesions were brown to gray, irregular to angular to circular, and 0.5 to 1 cm in diameter. Lesions on pods were oval to circular with reddish brown centers surrounded by darker brown borders. Conidia in vivo were curved cylindrical to obclavate with one to five septa and measured 25 to 60 × 3.5 to 7 µm. The conidiophores were 100 to 250 µm high and clustered together to form synnemata measuring 20 to 50 µm in diameter. The pathogen was isolated by placing conidia from diseased leaves onto potato dextrose agar (PDA) at pH 6. Colonies measuring 2 to 3 mm in diameter composed of dense, dark olive mycelium developed after incubation in the dark at 24 ± 2°C for 3 to 4 days. Pathogenicity of the isolate was tested with conidia obtained from the second subculture of 14-day-old colonies on PDA. Conidial suspensions of 2 × 104 conidia per ml were sprayed onto the upper and lower surfaces of the first trifoliolate leaves of six runner bean plants, 18 days after planting. Inoculated and control plants (sprayed with distilled water) were placed in a growth chamber with a 12-h photoperiod at 24 ± 2°C and 95 to 100% relative humidity and 48 h later moved to the greenhouse. Disease symptoms were evaluated 18 days after inoculation. While control plants were healthy, all inoculated plants showed symptoms similar to those observed in the field. The fungus that was consistently reisolated from lesions in the inoculated plants was identified as Phaeoisariopsis griseola on the basis of fungal morphology (1), symptoms produced on leaves (3), and random amplified polymorphic DNA data with primer 5'-CAATCGCCGT-3' (2). Runner bean is a new crop in Tafí del Valle, which is a geographically isolated area. In a period of only 2 years, the area cultivated with beans increased approximately five-fold. Because of this, the presence of a pathogen like Phaeoisariopsis griseola, which causes considerable reduction in yield in most common bean-producing areas of Argentina, is of concern. To our knowledge, this is the first report of ALS occurring on P. coccineus in Argentina. This report may prompt the inclusion of regular testing of seeds for ALS in P. coccineus-production areas. A voucher culture has been deposited in the LPSC (Culture collection of the La Plata Spegazzini Institute) No. 844. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, Surrey, UK, 1971. (2) P. Guzmán et al. Plant Dis. 83:37, 1999. (3) A. W. Saettler. Pages 15-16 in: Compendium of Bean Diseases. R. Hall, ed, The American Phytopathological Society, St. Paul, 1991. (4) S. A. Stenglein et al. Pages 209-243 in: Advances in Applied Microbiology, Vol. 52. A. I. Laskin et al., eds, Academic Press, San Diego, 2003.