A rice field owned by an individual grower in Lahore, Pakistan, was surveyed in July 2013. Plants with symptoms of black, circular, necrotic spots 3 to 4 mm in diameter and an average of 8 to 10 spots per leaf were observed. Diseased plants were present in the field either singly or in groups of three to five. Ten symptomatic plants were selected randomly, and one infected leaf per plant and one necrotic spot per leaf was selected for the isolation of the pathogen. Necrotic areas were cut into small pieces, surface sterilized with 1% sodium hypochlorite solution, and plated on 2% malt extract agar (MEA) (Sigma, Dorset, UK). After incubation at 25 ± 2°C for 4 to 5 days, fungal mycelium was transferred aseptically to fresh MEA for pure culture. Three different isolates grown for 7 days on MEA were selected for detailed morphological studies. The fungal colony was dark greenish-black, reaching 7 to 8 cm in diameter, with 2 to 3 poorly defined growth rings. Conidiophores were geniculate and 50 to 140 × 3 to 4.5 μm in size. Conidia were in chains of 4 to 10, ovoid, ranging in size from 35 to 50 × 8 to 10 μm, with 12 to 15 transversal and 0 to 2 longitudinal septa. Conidia darkened from dull tan yellow to brown as the culture aged. Based on morphological characteristics, the pathogen was identified as Alternaria gaisen (2). A pure culture of the pathogen was deposited in First Fungal Culture Bank of Pakistan (FCBP1354). Due to the complexity of morphology-based identification of the genus Alternaria, sequencing of the internal transcribed spacer (ITS) region was carried out using the ITS1/ITS4 primer pair (1,3). The nucleotide sequence (KJ806190) of an amplified DNA fragment was compared with those already submitted to GenBank. The BLAST results revealed 99% identity of our A. gaisen isolate to strains NW680 (EU520123.1), FC3s (JX391937.1), and CBS 632.93 (KC584197.1), as well as some other A. gaisen strains. Pathogenicity testing of the fungus was performed on Basmati-198, a common cultivar of rice in Pakistan, by either spraying leaves of 1-month-old plants with 10 ml of spore suspension (2 × 105 spores/ml) or mixing this spore suspension in soil at the time of sowing. Control plants were sprayed with sterilized water. Plants were kept in a glasshouse at 30 ± 2°C and monitored for disease development. After 15 days of incubation, similar leaf necrotic spots to those observed in the field, developed on all inoculated plants, whereas all control plants remained healthy and asympomatic. The experiment was repeated three times and similar results were obtained. Re-isolation of A. gaisen from the symptomatic leaves fulfilled Koch's pathogenicity postulate. Although limited to the field where it was observed, to our best of knowledge, this is the first report of rice leaf spot by A. gaisen from Pakistan. Also, rice has not been reported as the host of A. gaisen from any part of the world. This study indicates that A. gaisen is potentially an important pathogen of rice plants. Further investigations into epidemiology and disease management strategies for this new disease are warranted especially where rice crop is grown extensively.
References: (1) G. S. de Hoog and R. Horre. Mycoses 45:259, 2002. (2) E. G. Simmons. Alternaria: An identification manual. CBS, Fungal Biodiversity Center Utrecht, The Netherlands, 2007. (3) T. J. White et al. Page 315 in: PCR Protocols, A Guide to Methods and Applications, Academic Press, San Diego, 1990.
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