First Report of Phyllosticta aristolochiicola Causing Leaf Spot of Sonchus oleraceus in Pakistan.

During a survey of fields in and around the Quaid-e-Azam Campus, University of the Punjab, Lahore, Pakistan, during March 2015, leaves of sow thistle (Sonchus oleraceus), a plant having high medicinal and antimicrobial potential, infected with 1 to 4 mm necrotic dark brown irregular spots were observed. These spots coalesced forming larger necrotic areas. The same disease symptoms were recorded for theS. oleraceus plants growing in all the three sampling sites that were more than 100 meters away from each other. Approximately 70 to 80% leaf area and 100% of plants were infected with this disease. For pathogen isolation, leaves were collected from each sampling site. From each plant, 4 to 5 infected leaves and at least 3 to 4 necrotic spots per leaf were selected randomly, cut into approximately 3 mm2pieces, and surface sterilized with 1% sodium hypochlorite solution for 5 min. Five surface sterilized leaf pieces were inoculated onto malt extract agar (MEA) medium and incubated at 25 ± 2°C until fungal growth started from the inoculated leaf samples. Emerging hyphal tips were transferred to fresh MEA medium and incubated at 25 ± 2°C for purification. Cultural and morphological observations were made on 7-day-old pure fungus culture. Three purified isolates from three different sampling sites were selected randomly to identify the fungal pathogen. The colony of the isolated pathogen was black with irregular entire margins and slow growing, having a diameter of 3.7 cm. No sporulation was observed but abundant sterile dark brown branched and septate mycelia were present in 7-day-old culture. However, after 1 month of incubation at 25 ± 2°C, hyaline, subglobose conidia ranging from 9 to 15 × 7 to 10 µm were observed. For molecular characterization, total genomic DNA was obtained from one of the completely characterized isolate and used as template for the amplification of ITS region of rDNA by primer pair ITS1/ITS4 (White et al. 1990). The PCR product of approximately 650 bp was sent for sequencing. The resulting sequence was analyzed by BLAST showing 98% homology with Phyllosticta aristolochiicola strain BRIP 53316a having the GenBank ID NR_111791. Pure culture of P. aristolochiicola was deposited to FCBP and assigned accession FCBP1528 and ITS nucleotide sequence to GenBank under accession KT072733. To test pathogenicity, mycelium from 1-week-old pure fungus culture was suspended in Saline Tween 80 solution that was sprayed on three healthy plants followed by covering the each plant with polythene bag. Control plants were treated with sterilized distilled water and covered as the treated ones. All plants were kept at 27 ± 2°C and monitored regularly for the appearance of disease symptoms. Pathogenicity test was carried out three times using three different morphologically characterized isolates. Similar disease symptoms on leaves started to appear after 10 days of infection whereas control plants remained healthy. Reisolation of the same pathogen was confirmed by cultural characteristics as well as sequencing of the ITS1/ITS4 region, fulfilling Koch’s postulates. To our knowledge, this is the first report of P. aristolochiicola leaf spot ofS. oleraceus from Pakistan. Species of genus Phyllosticta are geographically widespread plant pathogens. The severity of disease observed at sampling sites was high; therefore, there is a need of proper management of this pathogen that might have a wider host range in Pakistan. © 2016 The American Phytopathological Society.