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Specific PCR-based detection of Alternaria helianthi: the cause of blight and leaf spot in sunflower

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Abstract

Alternaria helianthi is an important seed-borne pathogenic fungus responsible for blight disease in sunflower. The current detection methods, which are based on culture and morphological identification, are time-consuming, laborious and are not always reliable. A PCR-based diagnostic method was developed with species-specific primers designed based on the sequence data of a region consisting of the 5.8S RNA gene and internal transcribed spacers—ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of A. helianthi. The specificity of the primer pairs AhN1F and AhN1R designed was verified by PCR analysis of DNA from 18 Alternaria helianthi strains isolated from India, 14 non-target Alternaria spp. and 11 fungal isolates of other genera. A single amplification product of 357-bp was detected from DNA of A. helianthi isolates. No cross-reaction was observed with any of the other isolates tested. The detection limit of the PCR method was of 10 pg from template DNA. The primers could also detect the pathogen in infected sunflower seed. This species-specific PCR method provides a quick, simple, powerful and reliable alternative to conventional methods in the detection and identification of A. helianthi. This is the first report of an A. helianthi-specific primer set.

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Acknowledgments

The authors acknowledge the financial support and facilities provided by Danish Development Assistance (DANIDA), Copenhagen, Denmark, through Danish Seed Health Centre for the developing countries.

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Authors and Affiliations

  1. Department of Studies in Biotechnology, Asian Seed Health Centre, University of Mysore, Manasagangotri, Mysore, 570006, Karnataka, India

    A. C. Udayashankar, S. Chandra Nayaka, B. Archana, G. Anjana, S. R. Niranjana & H. S. Prakash

  2. Department of Agriculture and Ecology, Faculty of Life Sciences, Danish Seed Health Centre for Developing Countries, University of Copenhagen, Hoejbakkegaard Allé 30, 2630, Taastrup, Denmark

    C. N. Mortensen & Ole S. Lund

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  1. A. C. Udayashankar
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  2. S. Chandra Nayaka
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  3. B. Archana
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  4. G. Anjana
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  5. S. R. Niranjana
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  7. Ole S. Lund
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  8. H. S. Prakash
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Correspondence to H. S. Prakash.

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Communicated by Axal Brakhage.

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Udayashankar, A.C., Chandra Nayaka, S., Archana, B. et al. Specific PCR-based detection of Alternaria helianthi: the cause of blight and leaf spot in sunflower. Arch Microbiol 194, 923–932 (2012). https://doi.org/10.1007/s00203-012-0826-x

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  • DOI: https://doi.org/10.1007/s00203-012-0826-x

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