First Report of Leaf Spot of Rice Caused by Alternaria arborescens in Pakistan.

Rice (Oryza sativa) is one of the most profitable and popular cereal crops in Pakistan. In July 2012, symptoms consisting of circular, black, necrotic spots, 2 to 4 mm in diameter, were observed on leaves of a commonly grown rice cultivar, Basmati-198, in private rice fields at Lahore (Punjab). This disease was observed later on rice cultivar KSK-133 grown at Faisalabad (Punjab) during the same cropping season. Disease incidence was ~35% and 25% for Basmati-198 and KSK-133, respectively. To our knowledge, the pathogen was confined only in these areas and cultivars and was not present on other rice varieties or crops. Ten infected plants were selected randomly from each field of two rice cultivars and one infected leaf for each of the 10 infected plants was selected for the isolation of fungal pathogen. Necrotic lesions were cut into pieces of ~2 mm, surface-disinfected with 0.5% sodium hypochlorite, placed on 2% malt extract agar (MEA) (Sigma, Dorset, UK), and incubated at 25 ± 2°C for 4 to 5 days. Emerging fungal colonies were transferred aseptically to fresh MEA petri plates for purification. Alternaria spp. were consistently recovered from infected leaves. Three isolates per variety were selected for detailed morphological studies. Each isolate was grown at 25°C on MEA and potato carrot agar (PCA) for 7 days. All isolates displayed similar morphological features including black radiate, floccose colonies with irregular margins, 6 to 7 cm in diameter on MEA and 2 to 3 cm with 1 to 2 pairs of concentric growth rings on PCA. Conidial chains were not crowded with 1 to 10 conidia per branch and bearing several lateral branches. Conidiophores were tapering and narrow, 40 to 200 × 2 μm. Conidia were ovoid within a size range of 10 to 30 × 5 to 14 μm, with 1 to 5 transverse and 1 longitudinal septum. Conidial color darkens from a dull tan to a medium brown as the culture matures. Based on morphology, the pathogen was identified as Alternaria arborescens (1). A pure culture of the pathogen was deposited in First Fungal Culture Bank of Pakistan (FCBP) with accession FCBP1351. Identification based on morphology was verified by sequencing the internal transcribed spacer (ITS) region. For this, a DNA fragment of ~650 bp was amplified using total genomic DNA as template and ITS1 and ITS4 primers (2). The nucleotide sequence of the ITS region was submitted to GenBank under accession KF679683. Comparison of the sequence with those in GenBank revealed that the sequence was 99% identical with A. arborescens, isolate ALT-242 (KC415808), causing Eucalyptus leaf spot in India and strain STE-U4345 (AF404667), a causal pathogen of apple core rot in South Africa. Pathogenicity testing was performed on both cultivars. One-month-old plants grown in greenhouse were sprayed with 10 ml of spore suspension (2 × 10 spores/ml) as well as 10 ml of this spore suspension in soil at the time of sowing. Control plants were sprayed with sterilized water. The plants were covered with plastic bags for 48 h and kept under observation for 2 weeks in a glasshouse at 30 ± 2°C. Lesions appeared on leaves after 10 days of inoculation whereas control plants remained healthy. Pathogenicity tests were repeated in triplicate. Similar disease symptoms and re-isolation of A. arborescens fulfilled Koch's postulates. To our knowledge, this is the first report of A. arborescens leaf spot of rice in Pakistan. At present, the distribution of this disease is limited to the fields where it was observed. 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.