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Real-time PCR assay for rapid, efficient and accurate detection of Paramyrothecium roridum a leaf spot pathogen of Gossypium species

  • R. L. Chavhan
  • V. R. Hinge
  • U. S. Kadam
  • B. B. Kalbande
  • P. K. Chakrabarty
Original Article
  • 99 Downloads

Abstract

Here we report a highly sensitive real-time PCR (qPCR) assay to detect Paramyrothecium roridum from pure culture and infected samples of cotton plants. A specific set of primer pair pMyro F/R is designed to target the 185 bp ITS region of rDNA of Paramyrothecium roridum species and validated using qPCR. The fluorescence signals were detected above the baseline threshold from samples containing Paramyrothecium roridum DNA, whereas other samples did not produce any fluorescence or produced fluorescence which did not reach detection threshold values. A single dissociation peak of increased fluorescence was obtained for the specific primers at 92.2 °C melting temperature. The limit of detection using SYBR Green dye in this assay was up to 0.1 pg per µL of DNA from pure culture of P. roridum. The assay is accurate, sensitive, less laborious and time saving for detection of P. roridum in infected tissues of cotton.

Keywords

SYBR® Green Paramyrothecium roridum Leaf spot Cotton Internal transcribed spacer 

Notes

Acknowledgements

The authors gratefully acknowledge financial support under ICAR-Technology Mission on Cotton and DBT Grant No. BT/PR3900/GBD/27/386/2012.

Compliance with ethical standards

Conflict of interest

The authors declare that they do not have any conflict of interest.

References

  1. Appiah AA, Flood J, Archer SA, Bridge PD (2004) Molecular analysis of the major Phytophthora species on cocoa. Plant Pathol 53:209–219CrossRefGoogle Scholar
  2. Babu KB, Saxena AK, Srivastava AK, Arora DK (2007) Identification and detection of Macrophomina phaseolina by using species specific oligonucleotide primer and probe. Mycologia 99:797–803CrossRefGoogle Scholar
  3. Chakrabarty PK, Mayee CD (2004) Recent approaches to manage diseases of cotton. J Indian Soc Cotton Improv 29:65–79Google Scholar
  4. Chakrabarty PK, Chavhan RL, Sable S, Narwade AV, Monga D, Khadi BM (2007) Development of sensitive molecular diagnostic tools for detection of economically important fungal pathogens of cotton. In: World cotton conference 4, USAGoogle Scholar
  5. Chavhan RL, Hinge VR, Chinchole MB, Chakrabarty PK, Patade VY, Patil HB (2015) Rapid, specific and sensitive molecular detection assay for Alternaria helianthi that causes leaf blight disease in sunflower. Eur J Plant Pathol 143:663–675CrossRefGoogle Scholar
  6. Dake GN (1980) Effect of Myrothecium roridum on the germination of cotton seeds. Indian Phytopathol 33:591–593Google Scholar
  7. De Boer SH, Ward LJ, Li X, Chittaranjan S (1995) Attenuation of PCR inhibition in the presence of plant compounds by addition of BLOTTO. Nucleic Acid Res 23:2567–2568CrossRefPubMedPubMedCentralGoogle Scholar
  8. Demeke T, Jenkins GR (2010) Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits. Anal Bioanal Chem 396(6):1977–1990CrossRefPubMedGoogle Scholar
  9. Department of Agricultural Cooperation and Farmer’s Welfare, Annual Report, 2016–17. http://www.agricoop.nic.in/annual-report
  10. Grote D, Olmos A, Kofoet A, Tuset JJ, Bertolini E, Cambra M (2002) PCR detection of Pseudomonas humuli and Podosphaera macularis in Humulus lupulus. Plant Prot Sci 41(4):141–149Google Scholar
  11. Guillemette T, Iacomi-Vasilescu B, Simoneau P (2004) Conventional and real-time PCR based assay for detecting pathogenic Alternaria brassicae in cruciferous seed. Plant Dis 88:490–496CrossRefGoogle Scholar
  12. Hillock RJ (1992) Cotton diseases. CAB International, Redwood Press Ltd, Melksham, p 415Google Scholar
  13. 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 Sci Technol 30:87–95Google Scholar
  14. Jeeva ML, Mishra AK, Vidyadharan P, Misra RS, Hegde V (2010) A species-specific polymerase chain reaction assay for rapid and sensitive detection of Sclerotium rolfsii. Australas Plant Pathol 39(6):517–523CrossRefGoogle Scholar
  15. Jobes DV, Hurley DL, Thien LB (1995) Plant DNA isolation: a method to efficiently remove polyphenols, polysaccharides and RNA. Taxon 44:379–386CrossRefGoogle Scholar
  16. Kalbande BB, Patil AS, Chakrabarty PK (2016) An efficient, simple and high throughput protocol for cotton genomic DNA isolation. J Plant Biochem Biotechnol.  https://doi.org/10.1007/s13562-016-0360-z Google Scholar
  17. Kikuchi K, Matsushita N, Suzuki K (2007) Discrimination of Tricholoma species by species-specific ITS primers. Myco Sci 48:316–320CrossRefGoogle Scholar
  18. Lombard L, Houbraken J, Decock C, Samson RA, Meijer M, Réblová M, Groenewald JZ, Crous PW (2016) Generic hyper-diversity in Stachybo triaceae. Persoonia 36:156–246CrossRefPubMedPubMedCentralGoogle Scholar
  19. Martin RR, James D, Levesque CA (2000) Impacts of molecular diagnostic technologies on plant disease management. Annu Rev Phytopathol 38:207–239CrossRefPubMedGoogle Scholar
  20. Meyer MC, da Silva JC, Maia GL, Bueno CJ, Souza NL (2006) Mancha de mirotécioemalgodoeirocausadapor Myrothecium roridum. Summa Phytopathol 32(4):390–393CrossRefGoogle Scholar
  21. Mirmajlessi SM, Destefanis M, Gottsberger RA, Mänd M, Loit E (2015) PCR-based specific techniques used for detecting the most important pathogens on strawberry: a systematic review. Syst Rev 4(9):1–11Google Scholar
  22. Monga D (2011) Changing scenario of cotton diseases in India—the challenge ahead. In: World cotton conference, 2011. IndiaGoogle Scholar
  23. Nieves C, Ana MP, Ana A, Paloma ST (2012) Molecular tools for detection of plant pathogenic fungi and fungicide resistance. In: Plant pathology, Cumagun CJ (ed), ISBN: 978-953-51-0489-6Google Scholar
  24. Pavlina K, Peter JM, Marga PEG, Patricia VZ, Ruud VB (2002) Development of specific primers for detection and identification of Alternaria spp. in carrot material by PCR and comparison with blotter and plating assays. Mycol Res 106:23–33CrossRefGoogle Scholar
  25. Pavon MA, 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. J Food Prot 74:240–247CrossRefPubMedGoogle Scholar
  26. Pryor BM, Gilbertson RL (2001) A PCR-based assay for detection of Alternariara dicina on carrot seed. Plant Dis 85:18–23CrossRefGoogle Scholar
  27. Raut JG, Holey NR, Moghe PG (1980) Occurrence of Myrothecium leaf spot on cotton in Vidarbha. Indian Phytopathol 33:510–511Google Scholar
  28. Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols in the series Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365–386Google Scholar
  29. Srinivasan KV (1994) Cotton diseases. Published by Indian Society for Cotton Improvement. Pearl Lino Service, Thane, p 312Google Scholar
  30. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25(24):4876–4882CrossRefPubMedPubMedCentralGoogle Scholar
  31. Torres-Calzada C, Tapia-Tussell R, Quijano-Ramayo A, Martin-Mex R, Rojas-Herrera R, Higuera-Ciapara I, Perez-Brito D (2011) A species-specific polymerase chain reaction assay for rapid and sensitive detection of Colletotrichum capsici. Mol Biotechnol 49:48–55CrossRefPubMedGoogle Scholar
  32. Udayashankar AC, Chandra N, Archana B, Anjana G, Niranjana SR, Mortensen CN, Lund OS, Prakash HS (2012) Specific PCR-based detection of Alternaria helianthi: the cause of blight and leaf spot in sunflower. Arch Microbiol 194(11):923–932CrossRefPubMedGoogle Scholar
  33. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Shinsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego, pp 315–322Google Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2017

Authors and Affiliations

  • R. L. Chavhan
    • 1
  • V. R. Hinge
    • 1
  • U. S. Kadam
    • 1
  • B. B. Kalbande
    • 2
  • P. K. Chakrabarty
    • 2
    • 3
  1. 1.Vilasrao Deshmukh College of Agricultural Biotechnology (Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani)LaturIndia
  2. 2.ICAR-Central Institute for Cotton ResearchNagpurIndia
  3. 3.Plant Protection Section, Crop Science DivisionIndian Council of Agricultural Research, Krishi BhavanNew DelhiIndia

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