Characteristic Ascochyta blight lesions were observed on leaves and pods of wild pea (Pisum elatius Steven. ex M. Bieb.) growing at three sites in the Republic of Georgia during June and July of 2004. Site characteristics were 41°36.11′N, 44°31.34′E (elevation 919 m), 41°54.221′N, 44°05.667′E (elevation 744 m), and 41°44.907′N, 43°12.263′E (elevation 884 m). Lesions appeared similar to those induced by Ascochyta pisi Lib. on cultivated pea (P. sativum L.). Fungi were isolated by surface disinfesting small pieces of infected tissue in 95% EtOH for 10 s, 1% NaOCl for 1 min, and then in deionized sterile H20 for 1 min. Tissue pieces were placed on 3% water agar (WA) for 24 h under fluorescent lights with a 12-h photoperiod to induce sporulation. Single-conidial isolations were made by streaking conidia on 3% WA and picking germinated conidia 18 h later. Three fungi (isolates Georgia-6, -7, and -12) had colony morphology similar to that of A. pisi on V8 juice agar. Conidial suspensions (1 × 105 conidia/ml) of each isolate above were spray inoculated to runoff on three genotypes of 2-week-old P. elatius plants. Plants inoculated included PI lines 560055 and 513252 and W6 line 15006 from the USDA Western Region Plant Introduction Station, Pullman, WA with 11 replicate plants inoculated per isolate. Plants were incubated in a growth chamber for 48 h at 18°C and covered with a plastic cup to maintain high humidity. Characteristic Ascochyta blight lesions were apparent 7 days after inoculation. DNA was extracted from each isolate and 610 bp of the glyceraldehyde-3-phosphate-dehydrogenase gene (G3PD), 364 bp of chitin synthase 1, and 330 bp of the translation elongation factor 1-alpha gene were amplified with gpd-1 and gpd-2 primers (1), CHS-79 and CHS-354 primers (2), and EF1-728F and EF1-986R primers (2), respectively. Amplicons were direct sequenced on both strands, and BLAST searches of the NCBI nucleotide database with consensus G3PD, CHS, and EF sequences of isolates Georgia-6, -7, and -12 were performed. The closest match obtained for the G3PD sequences was A. pisi isolate ATCC 201617 (Accession No. DQ383963). G3PD sequences for Georgia-6, -7, and -12 were deposited in GenBank (Accession Nos. DQ383966 [Georgia-6 and -7] and DQ383963 [A. pisi isolate AP1 and Georgia-12]). Closest matches to CHS and EF sequences were A. pisi isolate ATCC 201618 (EF Accession No. DQ386494) and Didymella fabae isolate ATCC 96418 (CHS Accession No. DQ386481, EFAccession No. DQ386492), respectively. CHS sequences for Georgia-6, -7, and -12 were identical to each other and to A. fabae isolate AF1 and were deposited in GenBank (Accession No. DQ386481. EF sequences for Georgia-6, -7, and -12 were deposited in GenBank (Accession Nos. DQ386494 [Georgia-6 and A. pisi isolate AP2], DQ386495, and DQ386496, respectively. These results, coupled with the morphological identification and inoculation results, confirm the identity of the fungus as A. pisi. To our knowledge, this is the first report of Ascochyta blight of P. elatius in the Republic of Georgia.
References: (1) M. L. Berbee et al. Mycologia 91:964. 1999. (2) I. Carbone and L. M. Kohn. Mycologia 91:553, 1999.
