Potato (Solanum tuberosum) is one of the most important vegetable crops worldwide, including Pakistan. During surveys from November to February of 2011 to 2013 in Sahiwal (Punjab), a severe leaf spot disease, new to farmers, was recorded. Symptoms consisted of 1- to 3-mm diameter black circular necrotic spots and appeared on the leaves of 2- to 3-week-old plants. Disease incidence was ~70 to 80%. This disease was localized to few fields in Sahiwal on potato variety Sante and to our knowledge, this has not been found on other areas or potato varieties in Pakistan. Fungi were isolated from randomly selected diseased plants. Ten infected plants were brought to the laboratory in sterilized polyethylene bags. One infected leaf per plant was selected for pathogen isolation. Infected parts of leaves were cut into ~2 mm2 pieces. Leaf pieces were surface sterilized for 1 min with 0.5% sodium hypochlorite and then inoculated aseptically onto 2% malt extract agar (MEA) (Sigma, Dorset, UK) and incubated at 25 ± 2°C for 3 to 4 days in the dark. Hyphal tip transfer from emerging colonies was performed to obtain pure cultures. Initial microscopic examination of pure fungal colonies revealed Alternaria as the likely causal organism. For morphology-based identification, five isolates from separate infected leaves were grown on MEA as well as potato carrot agar (PCA) for 7 days. All isolates showed similar morphological characters including dusty greenish black, floccose colonies with regular and smooth margins reaching 3 to 4.5 cm in diameter on MEA and sporulation with well-defined zones of growth. Aerial hyphae produced long branches that bore lateral chains of 1 to 7 conidia. Conidia were pointed at the tip, ovoid or ellipsoid, ranged from 18 to 40 × 5 to 12 μm with 4 to 8 transverse and 0 to 1 longitudinal septa. No conidial beak was present. Conidial color darkened from dull olive to brown as the culture matured. Based on morphology, the pathogen was identified as Alternaria longipes (1). A pure culture of a fungal pathogen was submitted to First Fungal Culture Bank of Pakistan (FCBP1355) for future reference. To confirm the morphology-based identification, the rDNA internal transcribed spacer (ITS) nucleotide sequence was amplified using ITS1 forward and ITS4 reverse primers (2). The amplicon of 537 bp was sequenced and submitted to GenBank under accession KJ806191. A BLASTn search using the KJ806191 sequence revealed it to be 99% identical to around 20 different strains of A. longipes deposited in GenBank including leaf spot pathogens of another Solanaceaeous member, Nicotiana tabacum (AY154684) and Asteraceous plant, Atractylodes macrocepha (JQ004404). Pathogenicity testing was performed in the greenhouse at 30 ± 2°C. Pots (16 × 9 cm) were filled with sterilized soil. Since spores of Alternaria sp. are known to survive in soil or plant debris, soil was sterilized and inoculated with 106 spore suspension of the isolated pathogen before sowing the potato seeds. Control pots were not inoculated. Approximately 10 days after plant germination, the previously observed disease symptoms appeared on leaves and A. longipes was re-isolated from the necrotic areas of leaves, thus fulfilling Koch's postulates. Plants in control treatments were asymptomatic. Pathogenicity tests were repeated three times. To our knowledge, this is the first report of A. longipes leaf spot of potato cultivar Sante from Pakistan. However, the distribution of this disease is confined to the area where it was observed, but it could be a threat for potato crop if not managed timely.
References: (1) E. G. Simmons. Alternaria: An identification manual. CBS, Fungal Biodiversity Center Utrecht, The Netherlands, 2007. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.
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