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Poster: Diseases of Plants: Disease Detection & Diagnosis


Morphological and molecular identification of Phoma medicaginis var. medicaginis causing spring black stem and leaf spot of alfalfa in Nevada
R. Bomberger (1), S. Wang (1) (1) Nevada Department of Agriculture, U.S.A.

In June 2015, an outbreak of foliar blight disease was observed in an alfalfa (Medicago sativa L.) field with more than 50% of plants affected. Individual plants exhibited rapid death of foliage with sporadic or coalesced lesions on both lower leaves and stems. To isolate the causative agent, necrotic stem and leaf tissue, as well as pycnidia, were surface sterilized and then placed onto potato dextrose agar (PDA) amended with streptomycin sulfate and incubated at 22ÂșC in the dark. Colonies of Phoma species were obtained from all samples collected from 5 separate field locations. Ten single spore cultures (SSCs) were obtained from 10 isolates and compared morphologically. All SSCs and isolates produced almost black colonies with abundant hyaline, oval to cylindrical, single-celled conidia with no significant morphological variation observed. A 540 bp DNA fragment was amplified from the ITS regions of rRNA genes and it was sequenced directly from both strands. Sequence analysis revealed that all 10 SSCs had the same amplicon sequence (Accession No. KU695570), with 100% homology to the corresponding regions of P. medicaginis var. medicaginis (Accession No. AY504634). Thus, the pathogen isolated from diseased alfalfa plants in Nevada was determined to be P. medicaginis var. medicaginis, the only pathogen associated with the outbreak. This is the first confirmed outbreak of spring black stem disease in Nevada since 1977 when it was initially reported in the state.