INVESTIGADORES
GARRIZ andres
artículos
Título:
First report of Leptosphaeria biglobosa ´brassicae´ as causal agent of Phoma leaf spot in Brassica napus (Canola) in Argentina.
Autor/es:
ROSSI, FRANCO RUBÉN; ROMERO, FERNANDO MATÍAS; GÁRRIZ, ANDRÉS; RUÍZ, OSCAR ADOLFO
Revista:
PLANT DISEASE
Editorial:
AMER PHYTOPATHOLOGICAL SOC
Referencias:
Año: 2018
ISSN:
0191-2917
Resumen:
Canola (Brassica napus L.) is the second largest oilseed crop in the world providing 13% of the11 world?s oil supply. This crop has been grown in Argentina since the 1930s, and the area12 devoted to its cultivation varies every year, reaching a maximum of 95000 Ha in the 2012/201313 growing season.14 Phoma stem canker is considered the most important and devastating disease in Brassica15 napus and other Brassicae species [1]. The causal agent is a complex of two closely related16 fungal species, Leptosphaeria maculans and L. biglobosa. In Argentina, the presence of L.17 maculans in canola plants was reported for the first time in 2004 [2], but the existence of L.18 biglobosa has not been recorded so far.19 During the 2015/2016 season, we collected several leaf samples with typical Phoma leaf spot20 symptoms obtained from canola crops from the north and northeastern regions of the Buenos21 Aires province. The necrotic lesions were circular to irregularly oval, 8 to 15 mm in diameter,22 pale brown in the center, grayish green at the margin and characterized with the presence of23 pycnidia. Several leaf pieces with lesions were rinsed twice with deionized sterile water and24 placed in a humid chamber during 2-3 days to induce the pycnidia to exude cirri of conidia.25 After this period, one cirrus per sample was transferred onto PDA plates supplemented with26 antibiotics (15 mg/L STR, 15 mg/L GEN and 12 mg/L TET) using an inoculation needle under27 stereoscopic microscope. Thus, several isolates were obtained, some of them showing rapid28 mycelial growth rate and pigment production on PDA medium, as showed by the isolate29 Tapidor of L. biglobosa used as control (kindly provided by Professor Bruce Fitt, University of30 Hertfordshire-UK). Microscopic analysis showed hyaline conidias, subcylindrical with an31 average size of 4-5 x 1.5-2 μm [3]. In order to confirm the identity of these isolates, a PCR32 assay using genomic DNA as template was performed to distinguish L. maculans from L.33 biglobosa with the species-specific primers LmacR, LmacF, and LbigF in a three-primers34 strategy [4]. These reactions gave a 444-bp amplicon as expected for L. biglobosa ´brassicae´.35 In addition, these results were confirmed by sequencing the nuclear ribosomal internal36 transcribed spacer (ITS) region, which showed a 99% of identity with the sequence of L.37 biglobosa ´brassicae´ deposited at the GenBank database (FO905468). L. biglobosa isolates38 were then tested for pathogenicity on the canola cultivars Westar and Bioaureo 228639 (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 40 conidia/ml). Fourteen days41 after inoculation, irregular and brown necrotic lesions were visible at the site of inoculation.42 These cotyledons were detached and placed in a humid chamber to induce pycnidia formation.43 After three days cirri of conidia were transferred to a plate with PDA supplemented with44 antibiotics as mentioned above. The identity of these isolates of L. biglobosa were confirmed45 by pigment production on PDA medium and by PCR assay using species-specific primers. To46 our knowledge, this is the first report of L. biglobosa ´brassicae´ as a pathogen of canola in47 Argentina. This finding shows that not only L. maculans but also L. biglobosa are the causal48 agents of Phoma leaf spot in Argentina´s canola cropping areas. Therefore, precise molecular49 phenotyping techniques are necessaries to identify the causal agent of this disease.