Journal of Plant Diseases and Protection

, Volume 125, Issue 3, pp 331–337 | Cite as

Real-time assays for detection of Phytophthora spp. and identification of an avr3a gene variant

  • Shimaa R. T. Tolba
  • Mariella M. Finetti Sialer
  • Laura C. Rosso
  • Mahmoud M. A. Moustafa
  • Chiara Ruggeri
  • Ibrahim I. S. El-Shawaf
  • Aurelio CiancioEmail author
Short Communication


A study was carried out for identification and detection of Phytophthora spp. from soil and plant samples, collected from Solanaceous crops in Egypt and Italy. The samples were screened with specific and universal primers of Phytophthora 18S, ITS1–ITS2 and 28S regions, followed by PCR product sequencing. The Phytophthora spp. detected were P. infestans (Egypt) and P. parasitica (Italy). A molecular beacon probe was also developed based on the avr3a gene of P. infestans to detect a variant associated with virulence traits. The probe was suitable for avr3a allele identification from P. infestans and also from P. parasitica PCR products.


avr3a ITS Molecular beacon Phytophthora infestans Phytophthora parasitica Virulence 



Research developed as part of STT higher degree requirements. Study partially funded by the EU Commission, Joint Master Degree Tempus Project No. 543865. The authors thank prof. Sanaa Haroon (Fayum University, Egypt) for travel assistance and coordination, and Dr Michelina Ruocco, IPSP CNR, Portici, for suggestions and isolate CBS 120920.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Human and animal rights

Research does not involve human participants and/or animals.

Informed consent

All authors gave informed consent to publish the manuscript.


  1. Armstrong MR, Whisson SC, Pritchard L, Bos JIB et al (2005) An ancestral oomycete locus contains late blight avirulence gene Avr3a, encoding a protein that is recognized in the host cytoplasm. Proc Natl Acad Sci USA 102:7766–7771CrossRefPubMedPubMedCentralGoogle Scholar
  2. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidmann JG, Struhl K (1990) Current protocols in molecular biology, vol 3. Wiley, New York, p A.3–D.1Google Scholar
  3. Baka ZAM (1997) Mating type, nuclear DNA content and isozyme analysis of Egyptian isolates of Phytophthora infestans. Folia Microb 42:613–620CrossRefGoogle Scholar
  4. Beketova MP, Sokolova EA, Malyuchenko OP, Alekseev YI, Kuznetsova MA, Kozlovsky BE, Rogozina EV, Khavkin EE (2014) On molecular identification of Phytophthora infestans genotypes. Rus Agric Sci 40:435–438CrossRefGoogle Scholar
  5. Clément JAJ, Baldwin TK, Magalon H, Glais I, Gracianne C, Andrivon D, Jacquot E (2013) Specific detection and quantification of virulent/avirulent Phytophthora infestans isolates using a real-time PCR assay that targets polymorphisms of the Avr3a gene. Lett Appl Microb 56:322–332CrossRefGoogle Scholar
  6. Cooke DEL, Cano LM, Raffaele S, Bain RA, Cooke LR, Etherington GJ et al (2012) Genome analyses of an aggressive and invasive lineage of the Irish potato famine pathogen. PLoS Pathol 8:e1002940CrossRefGoogle Scholar
  7. Drenth A, Guest DI (2004) Principles of Phytophthora disease management. In: Drenth A, Guest DI (eds) Diversity and management of Phytophthora in Southeast Asia, ACIAR Monograph, vol 114. Australian Centre for International Agricultural Research, Canberra, Australia, pp 154–160Google Scholar
  8. El-Ganainy S, Ahmed Y, Soliman M, Ismail A, Tohamy A, Randall E, Cooke D (2016) A shift in the population of Phytophthora infestans on Egyptian potato crops. Phytopathology 106:140Google Scholar
  9. El-Korany AE (2008) Occurrence of oospores of Phytophthora infestans in the field and under controlled conditions. J Agric Environ Sci Alex Univ 7:31–52Google Scholar
  10. Ersek T, Schoelz JE, English JT (1994) PCR amplification of species-specific DNA sequences can distinguish among Phytophthora species. Appl Environ Microb 60:2616–2621Google Scholar
  11. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791CrossRefPubMedGoogle Scholar
  12. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95e98Google Scholar
  13. Judelson HS, Tooley PW (2000) Enhanced polymerase chain reaction methods for detecting and quantifying Phytophthora infestans in plants. Phytopathology 90:1112–1119CrossRefPubMedGoogle Scholar
  14. Jukes TH, Cantor CR (1969) Evolution of protein molecules. In: Munro HN (ed) Mammalian protein metabolism. Academic Press, New York, pp 21–132CrossRefGoogle Scholar
  15. Meszka B, Michalecka M (2016) Identification of Phytophthora spp. isolated from plants and soil samples on strawberry plantations in Poland. J Plant Dis Prot 123:29–36CrossRefGoogle Scholar
  16. Murry MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucl Acids Res 8:4321–4325CrossRefGoogle Scholar
  17. Ristaino JB (2002) Tracking historic migrations of the Irish potato famine pathogen, Phytophthora infestans. Microb Infect 4:1369–1377CrossRefGoogle Scholar
  18. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  19. Scibetta S, Schena L, Chimento A, Cacciola SO, Cooke DEL (2012) A molecular method to assess Phytophthora diversity in environmental samples. J Microb Methods 88:356–368CrossRefGoogle Scholar
  20. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefPubMedPubMedCentralGoogle Scholar
  21. 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. Nucl Acids Res 25:4876–4882CrossRefPubMedPubMedCentralGoogle Scholar
  22. Wilfinger WW, Mackey K, Chomczynski P (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques 22:474–478PubMedCrossRefGoogle Scholar

Copyright information

© Deutsche Phytomedizinische Gesellschaft 2018

Authors and Affiliations

  1. 1.Department of Genetics and Genetic Engineering, Faculty of AgricultureBenha UniversityMoshtohorEgypt
  2. 2.Consiglio Nazionale delle Ricerche, Istituto di Bioscienze e BiorisorseBariItaly
  3. 3.Consiglio Nazionale delle Ricerche, Istituto per la Protezione Sostenibile delle PianteBariItaly

Personalised recommendations