Characteristic Ascochyta blight lesions were observed on leaves and stems of pea (Pisum sativum L.) ‘Dove’ grown at two sites in the province of Burgos (northern Spain) during May and June of 2005 and 2006. Mean disease severity of affected tissue reached 47% in 2005 and 72% in 2006. Dark brown, circular, necrotic lesions were sometimes covered with pycnidia. Fungal isolations were made from small pieces of infected tissue by surface disinfecting in 1% NaOCl for 1 min and then washing in deionized, sterile water for 2 min. Tissue pieces were placed on potato dextrose agar (PDA) for 7 days at 20 to 24°C under fluorescents lights with a 12-h photoperiod to induce sporulation. Single-spore isolations were made by streaking conidia from PDA cultures on 2% water agar and picking germinated conidia after 18 h. Fungal colonies grown on PDA and conidia from these cultures were similar to that of Ascochyta pisi Lib., and no chlamydospores or pseudothecia were observed, eliminating the possibility that the isolated fungi were A. pinodes or A. pinodella (3), the other fungi associated with the “Ascochyta complex” of pea. Conidial suspensions (5 × 105 conidia/ml) of two single-spore isolates (Spain-47 and Spain-48) were spray inoculated to runoff on 3-week-old plants of bean (Phaseolus vulgaris L. ‘Contender’), chickpea (Cicer arietinum L. ‘Blanco lechoso’), lentil (Lens culinaris Medik. ‘Pardinar’), pea (‘Lincoln’), and faba bean (Vicia faba L. ‘Alameda’) with 10 replicate plants per isolate. Plants were incubated in a growth chamber at 20 to 24°C and 100% relative humidity (RH) for 48 h and then incubated at the same temperature and 50 to 80% RH for 3 weeks. Characteristic Ascochyta blight lesions were apparent 7 days after inoculation on leaves and stems of pea. No disease symptoms were observed on the other inoculated plants. DNA was extracted from both isolates (Spain-47 and Spain-48) and 610 bp of the glyceraldehyde-3-phosphate-dehydrogenase gene (G3PD) was amplified with gpd-1 and gpd-2 primers (2). Amplicons were direct sequenced on both strands and consensus sequences were aligned. Spain-47 and Spain-48 had identical sequences. A BLAST search of the NCBI nucleotide database with the consensus sequence revealed A. pisi G3PD Accession No. DQ383963 (isolate ATCC 201617, Bulgaria) as the closest match in the database with 100% sequence similarity. These results, coupled with the morphological identification and inoculation results, confirm the identity of the fungus as A. pisi. Although infections by A. pinodes or by unidentified Ascochyta spp. are well known in pea crops in Spain (1), to our knowledge, this is the first report of an outbreak of Ascochyta blight of pea caused by A. pisi under field conditions in Spain. References: (1) M. F. Andrés et al. Patógenos de Plantas Descritos en España. MEC, Madrid, 1998. (2) M. L. Berbee et al. Mycologia 91:964, 1999. (3) E. Punithalingam and P. Holliday. No 334 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1972.
Subcutaneous ticks: a first report in a golden jackal, and their absence in non-canid carnivores
