Indian Phytopath. 66 (3) : 263-268 (2013) In India, oilseed and vegetable crucifers viz., mustard, cauliflower, cabbage and broccoli are commonly grown which are affected by different fungal diseases amongst which leaf spot caused by Alternaria brassicae (Berk) Sacc and Alternaria brassicicola (Schwein) Wiltshire is a prevalent foliar disease widespread in all cruciferous crops (Meena et al., 2010) thus reducing the yield and quality. In vegetable Brassica seeds, A. brassicicola (Maude and Humpherson- Jones, 1980; Humpherson-Jones, 1985; Maude et al., 1984; Kubota et al., 2006) is the dominant pathogen, whereas in oilseed rape A. brassicae (Kolte, 1985; Humpherson-Jones and Phelps, 1989) is more common. Both the pathogens are seed borne in nature and dispersed by wind or rain during the growing season. For production of healthy seed, prevention of dark leaf spot in seed crops is a pre-requisite. The diagnostic leaf spot symptoms are quite similar for both A. brassicae and A. brassicicola therefore, it becomes difficult to distinguish them in a mixed infection, where confusion occurs for detecting the species of the pathogen. The conventional detection of pathogenic A. brassicae and A. brassicicola infection in crucifers including cultural, morphological and pathogenic characterization are time consuming and also need artificially controlled conditions which are temperature and humidity dependant. So true symptoms or exact detection is not possible between these two leaf spot pathogens in crucifers. In case of seed quarantine, crucifer seeds face serious difficulty as diagnosis is diffident because so many saprophytic Alternaria species are found on them, thus further the pathogen identification gets complicated. Polymerase chain reaction (PCR) based on amplification of internal transcribed spacer (ITS) region of the ribosomal DNA is one of the most sensitive and rapid method of fungal pathogen detection. Specific primer from non-ribosomal peptide synthase (NRPS) gene of A. brassicae were developed for detecting A. brassicae infection in crucifer seeds by conventional and real time PCR based assay (Guillemette et al., 2004). PCR based assay for A. brassicicola (Iacomi-Vasilescu et al., 2002) was demonstrated and the epidemiological and disease management aspects of A. brassicicola in India have been worked out but pathogen detection techniques need more extensive research as Indian isolates of A. brassicicola was not detectable through the pre existing detection protocols. The objective of the present study was to develop specific primer from the conserved ITS rDNA regions for the PCR based assay to detect Alternaria brassicicola in infected leaf and seed samples of cruciferous crops including both vegetables and oilseed crop. MATERIALS AND METHODS Isolation and collection of fungal cultures Alternaria infected leaf and stem samples from different cruciferous crops mainly cauliflower, cabbage, broccoli and mustard were collected from different parts of the country during 2009-2011. Cultures were grown and maintained on potato dextrose agar (PDA) plates and were identified on the basis of morphological characters. Some other species of pathogenic Alternaria viz., A. alternata (crucifers) and A. porri (onion) were also isolated. Fusarium oxysprorum f. sp. conglutinans and Aspergillus niger from crucifer seeds were isolated as seed contaminants. Preparation of seed macerates Seed samples were collected from the highly infected (naturally) fields of different cruciferous crops. Following crops PCR based assay for the detection of Alternaria brassicicola in crucifers PRATIBHA SHARMA*, SWATI DEEP, MANIKA SHARMA, DINESH SINGH BHATI and P. CHOWDAPPA 1 Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110 012, India 1 Indian Institute of Horticultural Research, Bengaluru 560 089, Karnataka, India ABSTRACT: Alternaria brassicicola (Schwein) Wiltshire and A. brassicae (Berk) Sacc. are important necrotrophic fungal pathogens causing black leaf spot disease on crucifers including vegetable and oilseed crops. In India, the mixed infection caused by both the species is a common phenomenon occurring in the field where the symptom becomes confusing and both the species become indistinguishable. The disease being seed borne requires an easy, fast and reliable detection method. A simple conventional polymerase chain reaction based assay was developed for the detection and identification of A. brassicicola using specific primers designed from nuclear ribosomal internal transcribed spacer (ITS) regions. A specific fragment of ~600bp was amplified by PCR using primers Acola-sens: 5′-GCA GCA TCT GCT GTT GGG G-3′ and Acola-reverse: 5′-CAA GGT CAG CAT CCA TAA AGC C-3′ for A. brassicicicola isolates, with a detection limit of 100pg. The primers did not support amplification when tested on A. brassicae, A. alternata, and A. porri. The primers could also detect seed-borne infection of A. brassicicola. Key words: Alternaria brassicicola, Crucifers, Internal transcribed spacer region (ITS), PCR assay RESEARCH ARTICLE *Corresponding author: psharma032003@yahoo.co.in