Abstract
The leaf blight disease of sunflower (Helianthus annuus) caused by Alternaria helianthi is a serious threat to its cultivation worldwide. Early and accurate detection of the pathogen is critical to efficient disease management and in avoiding further losses due to epidemics in sunflower. Conventional methods of detection and identification are time consuming, labour intensive, and lack specificity and sensitivity. A real-time PCR based TaqMan® probe assay was developed to target 156 bp Internal Transcribed Spacer (ITS) region of A. helianthi for its detection using fungal genomic DNA and infected plant tissues and seeds of sunflower. The specificity of the probe and primers was confirmed by testing their cross reactivity using genomic DNA of closely related Alternaria species isolated from 17 crop plants and 15 fungal species of other genera. No cross-reactivity could be detected with any of the other non-target fungal strains used in this study. The assay successfully detected as low as 1.0 pg fungal genomic DNA and up to 1 % infection in sunflower seed lots. To the best of our knowledge, this is the only probe based real-time PCR assay that enable high specificity and sensitivity for rapid detection of A. helianthi in infected seeds and plant tissues. The assay may also hold promise for application in effective bio-threat and risk mitigation program by early and accurate detection of the pathogen for effective management.
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Aroca, A., Raposo, R., & Lunello, P. (2008). A biomarker for the identification of four Phaeoacremonium species using the beta-tubulin gene as the target sequence. Applied Microbiology and Biotechnology, 80(6), 1131–1140.
Babu, B. K., Mesapogu, S., Sharma, A., Somasani, S. R., & Arora, D. K. (2011). Quantitative real-time PCR assay for rapid detection of plant and human pathogenic Macrophomina phaseolina from field and environmental samples. Mycologia, 103(3), 466–473.
Balasubramanyam, N., & Kolte, S. J. (1980). Effect of different intensities of Alternaria blight on yield and oil content of sunflower. Journal of Agricultural Science, 94, 749–751.
Bhargav, D. V., & Meena, H. P. (2014). Alternaria blight: a chronic disease in sunflower. Popular Kheti, 2(1), 146–153.
Broders, K. D., Woeste, K. E., San Miguel, P. J., Westerman, R. P., & Boland, G. J. (2011). Discovery of single-nucleotide polymorphisms (SNPs) in the uncharacterized genome of the ascomycete Ophiognomonia clavigignenti-juglandacearum from 454 sequence data. Molecular Ecology Resources, 11(4), 693–702.
Brouwershaven, I. V., Bruil, M. L., van Leeuwen, G. C. M., & Kox, L. F. F. (2010). A real-time (TaqMan) PCR assay to differentiate Monilinia fructicola from other brown rot fungi of fruit crops. Plant Pathology, 59, 548–555.
Capote, N., Pastrana, A. M., Aguado, A., & Sanchez, T. P. (2012). Molecular tools for detection of plant pathogenic fungi and fungicide resistance. In C. J. Cumagun (Eds.), Plant pathology (pp. 978–953). Agricultural and Biological Sciences.
Chander, R. (2003). Hybrid seed production Technology in Sunflower.Sunfower in India, DOR Hyderabad (ICAR), India.
Chavhan, R. L., Chakrabarty, P. K., Patil, F. S., Patil, H. B., Singh, S. K., & Khadi, B. M. (2008). Association of new fungal species with leaf spot and blight of sunflower and cloning of their ribosomal RNA genes. Indian Phytopathology, 61(1), 70–71.
Cho, M. S., Kang, M. J., Kim, C. K., Seol, Y. J., Hahn, J. H., Park, S. C., & Hwang, D. J. (2011). Detection of Xanthomonas oryzae pv. oryzae by real-time bio-PCR using pathovar-specific primers based on an rhs family gene. Plant Disease, 95, 589–594.
Dechassa, D., Rauscher, G., Koike, S. T., Mou, B., Hayes, R. J., Maruthachalam, K., Subbarao, K. V., & Klosterman, S. J. (2012). A real time PCR assay for detection and quantification of Verticillium dahlia in spinach seed. Phytopathology, 102, 443–451.
Dyer, P. S., Furneaux, P. A., Douhan, G., & Murray, T. D. (2001). A multiplex PCR test for determination of mating type applied to the plant pathogens Tapesia yallundae and Tapesia acuformis. Fungal Genetics and Biology, 33, 173–180.
Edel, V., Steinberg, C., Gautheron, N., & Alabouvette, C. (2000). Ribosomal DNA targeted oligonucleotide probe and PCR assay specific for Fusarium oxysporum. Mycological Research, 104, 518–526.
Garrido, C., Carbu, M., Fernández-Acero, F. J., Boonham, N., Colyer, A., Cantoral, J. M., & Budge, G. (2009). Development of protocols for detection of Colletotrichum acutatum and monitoring of strawberry anthracnose using real-time PCR. Plant Pathology, 58(1), 43–51.
Guillemette, T., Iacomi-Vasilescu, B., & Simoneau, P. (2004). Conventional and real-time PCR based assay for detecting pathogenic Alternaria brassicae in cruciferous seed. Plant Disease, 88, 490–496.
Hansford, C. G. (1943). Contributions towards the fungus flora of Uganda, V. Fungi Imperfecti. Proceedings of the Linnean Society of London, 154(1), 34–67.
Iacomi-Vasilescu, B., Blancard, D., Guenard, M., Molinero-Demilly, V., Laurent, E., & Simoneau, P. (2002). Development of a PCR based diagnostic assay for detecting pathogenic Alternaria species in cruciferous seeds. Seed Science and Technology, 30, 87–95.
Ioos, R., Fourrier, C., Iancu, G., & Gordon, T. (2009). Sensitive detection of Fusarium circinatum in pine seed by combining an enrichment procedure with a real time polymerase chain reaction using dual-labeled probe chemistry. Phytopathology, 99(5), 582–590.
Jasalavich, C. A., Morales, V. M., Pelcher, L. E., & Seguin, S. G. (1995). Comparison of nuclear ribosomal DNA sequences from Alternaria species pathogenic to crucifers. Mycological Research, 99, 604–614.
Kusaba, M., & Tsuge, T. (1995). Phylogeny of Alternaria fungi known to produce host-specific toxins on the basis of variation in internal transcribed spacers of ribosomal DNA. Current Genetics, 28, 491–498.
Levesque, C. A. (2001). Molecular methods for detection of plant pathogens what is the future. Canadian Journal Plant Pathology, 23, 333–336.
Lievens, B., Van Baarlen, P., Verreth, C., VanKerckhove, S., Rep, M., & Thomma, B. P. (2009). Evolutionary relationships between Fusarium oxysporum f. splycopersici and F. oxysporum f. spradicis-lycopersici isolates inferred from mating type, elongation factor-1 alpha and exopolygalacturonase sequences. Mycological Research, 113, 1181–1191.
Martin, F. N., & Tooley, P. W. (2003). Phylogenetic relationships among Phytophthora species inferred from sequence analysis of mitochondrially encoded cytochrome oxidase I and II genes. Mycologia, 95(2), 269–284.
Mostert, L., Groenewald, J. Z., Summerbell, R. C., Gams, W., & Crous, P. W. (2006). Taxonomy and pathology of Togninia (Diaporthales) and its Phaeoacremonium anamorphs. Studies in Mycology, 54, 1–113. doi:10.1094/PHYTO-96-0336.
Mule, G., Susca, A., Logrieco, A., Stea, G., & Visconti, A. (2006). Development of a quantitative real-time PCR assay for the detection of Aspergillus carbonarius in grapes. International Journal of Food Microbiology, 111, 28–34.
Narian, U., & Chauhan, L. S. (1981). Leaf spot of sunflower caused by A. tenuissima in India. FAO Plant Protection Bulletin, 29(1/2), 29.
Nguyen, H. D. T., & Seifert, K. A. (2008). Description and DNA barcoding of three new species of Leohumicola from South Africa and the United States. Persoonia, 21, 57–69.
Pachkhede, A. U., Vyawahare, S. Y., & Shreemali, L. (1985). Two new species of Phoma from India. Current Science, 54, 485–486.
Pavlina, K., Marga, P. E., Van, G., Patricia, V. D. Z., & Bulk, R. D. (2002). Development of specific primers for detection and identification of Alternaria spp. in carrot material by PCR and comparison with blotter and plating assays. Mycological Research, 106(1), 23–33.
Pavon, M. A., Gonzalez, I., Rojas, M., Pegels, N., Martin, R., & Garcia, T. (2011). PCR detection of Alternaria spp. in processed foods, based on the internal transcribed spacer genetic marker. Journal of Food Protection, 74, 240–247.
Pavon, M., González, I., Martín, R., & Garcialacarra, T. (2012). ITS-based detection and quantification of Alternaria spp. in raw and processed vegetables by real-time quantitative PCR. Food Microbiology, 32(1), 165–171.
Prasad, M. S. L., Sujatha, K., & Rao, S. C. (2010). Seed transmission of Alternaria helianthi, incitant of leaf blight of sunflower. Journal of Mycology and Plant Pathology, 40, 63–66.
Pryor, B. M., & Gilbertson, R. L. (2001). A PCR-based assay for detection of Alternaria radicina on carrot seed. Plant Disease, 85, 18–23.
Rao, S., & Ramgopal, S. (2010). Effect of Alternaria helianthi culture filtrate on callus and regeneration of plantlets from tolerantcallus in sunflower. Indian Journal of Biotechnology, 9, 187–191.
Raut, J. G. (1985). Location of Alternaria helianthi in the sunflower seeds and its transmission from seed to plant. Indian Phytopathology, 38, 522.
Sarlin, T., Tapani, Y., Marika, J., Aldo, R., & Sari, P. (2006). Real-time PCR for quantification of toxigenic Fusarium spp. in barley and malt. European Journal of Plant Pathology, 114, 371–380.
Sawant, K. L. (1989). Invstigation on the destructive blight disease of sunflower. M.Sc.(Agri) Thesis M.A.U. Parbhani, pp. 74.
Seifert, K. A., Samson, R. A., Dewaard, J. R., Houbraken, J., Levesque, C. A., Moncalvo, J. M., Louis-Seize, G., & Hebert, P. D. N. (2007). Prospects for fungus identification using CO1 DNA barcodes, with Penicillium as a test case. Proceedings of the National Academy of Sciences of the United States of America, 104(10), 3901–3906.
Simmons, E. G. (2007). Alternaria: an identification manual. Centralbureau voor Schimmelcultures, Utrecht, Netherlands. CBS Biodiversity Series, 6, 667–668.
Steve, R., & Helen, J. S. (2000). Primer3 on the WWW for general users and for biologist programmers. In S. Krawetz & S. Misener (Eds.), Bioinformatics methods and protocols: Methods in molecular biology (pp. 365–386). Totowa: Humana Press.
Thompson, J. D., Higgins, D. G., & Gibson, T. J. (1994). Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, 4673–4680.
Tooley, P. W., Martin, F. N., Carras, M. M., & Frederick, R. D. (2006). Real-time fluorescent PCR detection of Phytophthora ramorum and Phytophthora pseudosyringae using mitochondrial gene regions. Phytopathology, 96, 336–345.
Udayashankar, A. C., Chandra, N., Archana, B., Anjana, G., Niranjana, S. R., Mortensen, C. N., Lund Ole, S. H., & Prakash, S. (2012). Specific PCR-based detection of Alternaria helianthi: the cause of blight and leaf spot in sunflower. Archives of Microbiology, 194(11), 923–932.
Weising, K., Nybom, H., Wolff, K., & Meyer, W. (1995). DNA isolation and purification. In K. Weising (Ed.), DNA fingerprinting in plants and fungi (pp. 51–54). Boca Raton: CRC Press.
White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M. A. Innis, D. H. Gelfand, J. J. Shinsky, & T. J. White (Eds.), PCR protocols: A guide to methods and applications (pp. 315–322). San Diego: Academic.
Xue, B., Goodwin, P. H., & Aninis, S. L. (1992). Pathotype identification of Leptosphaeria maculans I with PCR of oligonucleotide primers from ribosomal internal transcribed spacer sequences. Molecular Plant Pathology, 141, 179–188.
Yadav, M. K., Babu, B. K., Saxena, A. K., Singh, B. P., & Singh, K. (2011). Real-time PCR assay based on Topoisomerase II gene for detection of Fusarium udum. Mycopathologia, 171, 373–381.
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Financial grant offered by the Department of Biotechnology (DBT), Government of India, New Delhi, to support this research is gratefully acknowledged.
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Chavhan, R.L., Hinge, V.R., Chinchole, M.B. et al. Rapid, specific and sensitive molecular detection assay for Alternaria helianthi that causes leaf blight disease in sunflower. Eur J Plant Pathol 143, 663–675 (2015). https://doi.org/10.1007/s10658-015-0716-6
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DOI: https://doi.org/10.1007/s10658-015-0716-6