ROMERO Fernando Matias
First report of Leptosphaeria biglobosa ´brassicae´ as causal agent of Phoma leaf spot in Brassica napus (Canola) in Argentina.
ROSSI, FRANCO RUBÉN; ROMERO, FERNANDO MATÍAS; GÁRRIZ, ANDRÉS; RUÍZ, OSCAR ADOLFO
AMER PHYTOPATHOLOGICAL SOC
anola (Brassica napus L.) is the second largest oilseed crop in the world providing 13% of the world?s oil supply. This crop has been grown in Argentina since the 1930s, and the area devoted to its cultivation varies every year, reaching a maximum of 95000 Ha in the 2012/2013 growing season. Because of the occurrence of optimal weather conditions and soils with high fertility, the average yield in this region is about 2000 kg/Ha. Phoma leaf spot and Phoma stem canker are considered the most important and devastating diseases in Brassica napus and other Brassicae species . In both cases, the causal agent is a complex of two closely related fungal species, Leptosphaeria maculans and L. biglobosa. In Argentina, the presence of L. maculans in canola plants was reported for the first time in 2004 , but the existence of L. biglobosa has not been recorded so far. During the 2015/2016 season, we collected several samples with typical Phoma leaf spot symptoms from canola plants growing in fields from the north and northeastern regions of the Buenos Aires province. The necrotic lesions were circular to irregularly oval, 8 to 15 mm in diameter, pale brown in the center, grayish green at the margin and characterized with the presence of pycnidia. Several leaf pieces with lesions were rinsed twice with deionized sterile water and placed in a humid chamber (90 mm diameter Petri dish with a layer of filter paper soaked in deionized sterile water) during 2-3 days to induce the pycnidia to exude cirri of conidia. After this period, one cirrus per sample was transferred onto PDA plates supplemented with antibiotics (15 mg/L streptomycin, 15 mg/L gentamicin and 12 mg/L tetracycline) using an inoculation needle under stereoscopic microscope. Thus, several isolates were obtained, some of them showing rapid mycelial growth rate and pigment production on PDA medium, as showed by the isolate Tapidor of L. biglobosa that we used as control (kindly provided by Professor Bruce Fitt, University of Hertfordshire-UK). In order to confirm the identity of these isolates, a PCR assay using genomic DNA as template was performed to distinguish L. maculans from L. biglobosa with the species-specific primers LmacR, LmacF, and LbigF in a three-primers strategy described by Gaetán (2005). These reactions gave a 444-bp amplicon as expected for L. biglobosa ´brassicae´. In addition, these results were confirmed by sequencing the nuclear ribosomal internal transcribed spacer (ITS) region, which showed a 99% of identity with the sequence of L. biglobosa ´brassicae´ at the GenBank database (FO905468). L. biglobosa isolates were then tested for pathogenicity on the canola cultivars Westar and Bioaureo 2286 (Nuseed). With this purpose, cotyledons of 10-day-old seedlings were pricked with a needle, and each wound inoculated with 10 μl of a conidial suspension (107 conidia/ml). Sterilized distilled water was used as control. Developing primary leaves were removed every 2-3 days in order to ensure that cotyledons continue to expand. Fourteen days after inoculation, irregular and brown necrotic lesions were visible at the site of inoculation. These cotyledons were detached and placed in a humid chamber to induce pycnidia formation. After three days cirri of conidia were transferred to a plate with PDA supplemented with antibiotics as mentioned above. The identity of these isolates of L. biglobosa were confirmed by pigment production on PDA medium and by PCR assay using species-specific primers. To our knowledge, this is the first report of L. biglobosa ´brassicae´ as a pathogen of canola in Argentina. This finding shows that in Argentina´s canola cropping areas not only L. maculans but also L. biglobosa are the causal agents of Phoma leaf spot disease.