Lesions were observed on leaves and stems of alfalfa (Medicago sativa L.) growing as weeds in Pullman, Washington in June of 2001. Lesions appeared similar to those described for spring black stem and leaf spot caused by Phoma medicaginis Malbr. & Roum. in Roum. var. medicaginis Boerema (synonyms Phoma herbarum Westend. var. medicaginis Fckl. and Ascochyta imperfecta Peck). Sporulation was induced by placing surface-disinfested pieces of infected tissue on 3% water agar (WA) for 24 h under fluorescent light with a 12-h photoperiod. Single-conidial isolations were made by streaking conidia on 3% WA and picking germinated conidia after 18 h. Isolates had cultural and conidial morphology similar to descriptions of P. medicaginis and isolate ATCC52798 when grown on V8 agar and PDA at room temperature (3). Distinction between P. medicaginis var. medicaginis and P. medicaginis var. macrospora was not attempted. Conidial suspensions (1 × 106 conidia/ml) of isolates AS1, AS2, AS3, and AS4 were spray inoculated to runoff onto 3-week-old plants. PI lines 536535 and 536534 of M. sativa subsp. sativa (4-trifolate stage) and PI lines 442896 and 577609 of M. truncatula (5- to 7-trifolate stage) from the USDA Western Region Plant Introduction Station, Pullman, Washington were inoculated, with at least two replicate plants inoculated per isolate. Plants were incubated in a dew chamber at 20°C in the dark for 24 h to promote infection and then transferred to a growth chamber at 18°C with a 12-h photoperiod. Lesions were apparent on M. sativa subsp. sativa plants 4 days postinoculation (dpi) and 7 dpi on M. truncatula plants. At 12 dpi, many dark brown lesions with chlorotic halos were noted on leaves of M. sativa subsp. sativa, occasionally killing the entire trifoliate leaf and progressing approximately 1 cm down the stem. According to the previously published 1-to-5 visual rating scale for this disease (4), disease scores on both genotypes of M. sativa subsp. sativa were 4 (susceptible), while disease ratings on M. truncatula were 1-2 (resistant) with a few dark brown lesions noted on leaves and stems generally restricted to less than 2 mm in diameter. DNA was extracted from isolates AS1 and AS4, and PCR was performed using gpd-1 and gpd-2 primers for the glyceraldehyde-3-phosphate dehydrogenase gene (G3PD) (1), and EF1-728F and EF1-986R primers for the translation elongation factor 1-alpha gene (EF) (2), resulting in amplification of an approximately 600-bp fragment from each primer set. Amplicons were direct-sequenced on both strands, and BLAST searches of the NCBI nucleotide database were conducted with consensus G3PD and EF sequences of both isolates AS1 and AS4. Closest matches obtained for the G3PD and EF sequences were P. medicaginis isolate ATCC52798 (Accession No. DQ525740) and P. medicaginis var. medicaginis CBS316.90 (Accession No. AY831548), respectively. The G3PD and EF sequences for these isolates have been deposited in GenBank database (Accession Nos. EU394712--EU394715). To our knowledge, this is the first confirmed report of spring black stem and leaf spot of alfalfa in Washington State supported by Koch's postulates, cultural morphology, and multigene sequencing.
References: (1) M. L. Berbee et al. Mycologia 91:964, 1999. (2) I. Carbone and L. M. Kohn. Mycologia 91:553, 1999. (3) G. C. Kinsey. No. 1503 in: IMI Descriptions of Fungi and Bacteria. CABI Bioscience, Surrey, UK, 2002. (4) R. M. Salter and K. L. Leath. Spring blackstem and leafspot resistance. Online publication. North American Alfalfa Improvement Conference, Beltsville, MD, 1992.
