FIRST REPORT OF CLADOSPORIUM OXYSPORUM CAUSING LEAF SPOT OF ROSA INDICA IN PAKISTAN
N. Akhtar, M. Nayab
In December 2014, necrotic leaf spots were observed on more than fifty rose (Rosa indica) plants in a private garden at Lahore, Pakistan, all showing the same symptoms The infected plants were 1-2 year old and on an average 90% of the leaves were covered with 2-5 mm circular black spots, with irregu- lar margins without chlorosis around the margins. Initially, infected leaves remained attached to the plants but dropped when the spots coalesced and covered more than 80% leaf area. No symptoms were observed on flowers. For pathogen isolation, one spot per leaf from six symptomatic leaves of dif- ferent plants were cut into pieces, surface sterilized, inoculated on 2% malt extract agar (MEA) and incubated at 25 ± 2oC. Morphological observations were made on 7-day-old pure cul- tures. Colonies were greenish black and attained the diameter of 4 mm. Conidiophores were macronematous, smooth walled, up to 500μm long and 4-5μm wide, with intercalary and termi- nal swellings. Conidia were pale brown arising from terminal swelling of conidiophores, in the form of simple or branched chains. Conidia were spherical, subspherical or limoniform, 3-6 μm in diameter; ramoconidia were 2-4×7-3 μm in size. A representative pure culture of the fungus was deposited to First Fungal Culture Bank of Pakistan under the accession No.FCBP1517. Based on morphology, the fungus was identi- fied as Cladosporium oxysporum (Bensch et al., 2010). For se- quencing of ITS region, a DNA fragment of ca. 600 bp was amplified using universal primer pair ITS1/ITS4 and total ge- nomic DNA as template. BLASTn results indicated that ITS nucleotide sequence of this strain (KT283681) had 99% simi- larity with many other isolates of C. oxysporum in GenBank, including KT936546, LC040920, KJ475816 and JQ775499. Pathogenicity test was performed three times on young pot- ted rose plants using two different isolates. Since the inoculum of this fungus persists in soil, conidia from pure cultures were suspended in sterilized water (107 spores/ml) and sprayed in the soil of three healthy plants; three control plants were treated similarly with sterilized water. All plants, covered with poly- thene bags, were kept in a growth chamber at 23 ± 2oC. Ne- crotic spots similar to those described above started appearing on leaves only on inoculated plants after 21 days of incubation. Re-isolation of same fungus fulfilled Koch’s postulates. To our knowledge, this is the first report of R. indica leaf spot caused by C. oxysporum in Pakistan.