Plant and Soil

, Volume 443, Issue 1–2, pp 605–612 | Cite as

Improved real-time PCR protocol for the accurate detection and quantification of Rosellinia necatrix in avocado orchards

  • Juan M. Arjona-López
  • Nieves Capote
  • Carlos J. López-HerreraEmail author
Methods Paper



This study aims to develop and validate a new molecular method of detection and quantification of Rosellinia necatrix fungus in soil samples and compare it with conventional methods.


We collected 40 soil and root samples (one as negative control) from the soil around avocado trees. The root samples were checked for typical symptoms of R. necatrix and the pathogen was identified using the conventional method of plate culture. These results were then corroborated using a new molecular method of detection and quantification of R. necatrix in soil samples, and a duplex TaqMan qPCR protocol was designed that included an internal positive control to avoid the detection of false negatives.


The molecular detection and quantification method was effective, sensitive and reliable for all 40 soil samples analysed, whereas, with traditional methods, the fungus was isolated in only 24 out of the 26 symptomatic roots from 40 avocado trees sampled. This improved methodology reduces the sample preparation time compared with previous studies, and provides a molecular tool for the reliable and accurate detection and quantification of R. necatrix in naturally infested avocado soils.


This technique could be applied for the rapid assessment of R. necatrix in soils at the pre-planting stage and evaluation of the efficacy of physical, chemical or biological control treatments.


qPCR TaqMan R. necatrix Quantification Soil 



This study was partly supported by the Spanish Plan Nacional I + D + I Ministerio de Economía y Competitividad (AGL 2014-52518-C2-2-R). The research was also co-financed by FEDER funds (EU). The authors would like to thank TROPS for their technical support, especially in the location of isolates.


  1. Agustí-Brisach C, Mostert L, Armengol J (2014) Detection and quantification of Ilyonectria spp. associated with black foot disease of grapevine in nursery soils using multiplex nested PCR and quantitative PCR. Plant Pathol 63:316–322. CrossRefGoogle Scholar
  2. Anselmi N, Giorcelli A (1990) Factors influencing the incidence of Rosellinia necatrix Prill. in poplars. For Pathol 20:175–183. CrossRefGoogle Scholar
  3. Arakawa M, Nakamura H, Uetake Y, Matsumoto N (2002) Presence and distribution of double-stranded RNA elements in the white root rot fungus Rosellinia necatrix. Mycoscience 43:21–26. CrossRefGoogle Scholar
  4. Arjona-Lopez JM, Telengech P, Jamal A et al (2018) Novel, diverse RNA viruses from Mediterranean isolates of the phytopathogenic fungus, Rosellinia necatrix: insights into evolutionary biology of fungal viruses. Environ Microbiol 20:1464–1483. CrossRefPubMedGoogle Scholar
  5. Bilodeau GJ, Koike ST, Uribe P, Martin FN (2012) Development of an assay for rapid detection and quantification of Verticillium dahliae in soil. Phytopathology 102:331–343. CrossRefPubMedGoogle Scholar
  6. Capote N, Pastrana AM, Aguado A, Sánchez-Torres P (2012) Molecular tools for detection of plant pathogenic fungi and fungicide resistance.  In: Cumagun, C.J.(ed), Plant Pathology. InTech, Rijeka, Croatia, pp 151−202. Available from:−pathology/molecular-tools-for-detection−of−plant−pathogenic−fungi−and−fungicide−resistance. Accessed 15 February 2019Google Scholar
  7. de la Lastra E, Basallote−Ureba MJ, De los Santos B et al (2018) A TaqMan real−time polymerase chain reaction assay for accurate detection and quantification of Fusarium solani in strawberry plants and soil. Sci Hortic 237:128−134.
  8. de Mendiburu F (2013) Statistical procedures for agricultural research. Package “Agricolae”, version 1.4−4. Comprehensive R Archive Network, Institute for Statistics and Mathematics, ViennaGoogle Scholar
  9. Delatour C, Guillaumin JJ (1985) Importance des pourridies dans les régions tempérées. Eur J For Pathol 15:258–263. CrossRefGoogle Scholar
  10. Eguchi N, Kondo K, Yamagishi N (2009) Bait twig method for soil detection of Rosellinia necatrix, causal agent of white root rot of Japanese pear and apple, at an early stage of tree infection. J Gen Plant Pathol 75:325–330. CrossRefGoogle Scholar
  11. Francis SM (1985) Rosellinia necatrix–fact or fiction. Sydowia 38:75–86Google Scholar
  12. Kageyama K, Komatsu T, Suga H (2003) Refined PCR protocol for detection of plant pathogens in soil. J Gen Plant Pathol 69:153–160. CrossRefGoogle Scholar
  13. López−Herrera CJ (1989) Podredumbres radiculares del aguacate en la Costa del Sol. Años 1987−88. In: Estudios Fitopatología. J. Moral (ed), SEF/DGIEA Badajoz, pp 172−176Google Scholar
  14. López-Herrera CJ (2000) Podredumbre blanca de la raíz causada por Rosellinia necatrix. In: Enfermedades de los frutales de pepita y hueso. Mundi-Prensa (ed), Madrid, pp 79–81Google Scholar
  15. López-Herrera CJ, Zea-Bonilla T (2007) Effects of benomyl, carbendazim, fluazinam and thiophanate methyl on white root rot of avocado. Crop Prot 26:1186–1192. CrossRefGoogle Scholar
  16. López−Herrera CJ, Perez-Jimenez RM, Basallote-Ureba MJ et al (1997) Effect of soil solarization on the control of Phytophthora root rot in avocado. Plant Pathol 46:329–340.
  17. López-Herrera CJ, Pérez-Jiménez RM, Zea-Bonilla T et al (1998) Soil Solarization in established avocado trees for control of Dematophora necatrix. Plant Dis 82:1088–1092. CrossRefPubMedGoogle Scholar
  18. Mahuku G (2000) Detection methods for Verticillium species in naturally infested and inoculated soils. Am J Potato Res 77:271–274. CrossRefGoogle Scholar
  19. MAPAMA (2019) Accessed 6 February 19
  20. Pasini L, Prodorutti D, Pastorelli S, Pertot I (2016) Genetic diversity and biocontrol of Rosellinia necatrix infecting apple in northern Italy. Plant Dis 100:444–452. CrossRefPubMedGoogle Scholar
  21. Pérez-Artés E, Mercado-Blanco J, Ruz-Carrillo AR et al (2005) Detection of the defoliating and nondefoliating pathotypes of Verticillium dahliae in artificial and natural soils by nested PCR. Plant Soil 268:349–356. CrossRefGoogle Scholar
  22. Pliego C, Kanematsu S, Ruano-Rosa D et al (2009) GFP sheds light on the infection process of avocado roots by Rosellinia necatrix. Fungal Genet Biol 46:137–145. CrossRefPubMedGoogle Scholar
  23. Pliego C, Ramos C, de Vicente A, Cazorla FM (2011) Screening for candidate bacterial biocontrol agents against soilborne fungal plant pathogens. Plant Soil 340:505–520. CrossRefGoogle Scholar
  24. Prigigallo MI, Mosca S, Cacciola SO et al (2015) Molecular analysis of Phytophthora diversity in nursery-grown ornamental and fruit plants. Plant Pathol 64:1308–1319. CrossRefGoogle Scholar
  25. Ruano-Rosa D, Schena L, Ippolito A, López-Herrera CJ (2007) Comparison of conventional and molecular methods for the detection of Rosellinia necatrix in avocado orchards in southern Spain. Plant Pathol 56:251–256. CrossRefGoogle Scholar
  26. Ruano-Rosa D, Arjona-Girona I, López-Herrera CJ (2017) Integrated control of avocado white root rot combining low concentrations of fluazinam and Trichoderma spp. Crop Prot.
  27. Schena L, Ippolito A (2003) Rapid and sensitive detection of Rosellinia necatrix in roots and soils by real time scorpion-PCR [woody plants−Italy]. J Plant Pathol (Italy).Google Scholar
  28. Schena L, Nigro F, Ippolito A (2002) Identification and detection of Rosellinia necatrix by conventional and real-time scorpion-PCR. Eur J Plant Pathol 108:355–366. CrossRefGoogle Scholar
  29. Schena L, Nigro F, Ippolito A (2004a) Real-time PCR detection and quantification of soilborne fungal pathogens: the case of Rosellinia necatrix, Phytophthora nicotianae, P. citrophthora, and Verticillium dahliae. Phytopathol Mediterr 43:273–280Google Scholar
  30. Schena L, Nigro F, Ippolito A, Gallitelli D (2004b) Real-time quantitative PCR: a new technology to detect and study phytopathogenic and antagonistic fungi. Eur J Plant Pathol 110:893–908. CrossRefGoogle Scholar
  31. Shishido M, Kubota I, Nakamura H (2012) Development of real-time PCR assay using TaqMan probe for detection and quantification of Rosellinia necatrix in plant and soil. J Gen Plant Pathol 78:115–120. CrossRefGoogle Scholar
  32. Sztejnberg A, Madar Z (1980) Host range of Dematophora necatrix, the cause of white root rot disease in fruit trees. Plant Dis 64:662–664. CrossRefGoogle Scholar
  33. ten Hoopen GM, Krauss U (2006) Biology and control of Rosellinia bunodes, Rosellinia necatrix and Rosellinia pepo: a review. Crop Prot 25:89–107. CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Juan M. Arjona-López
    • 1
  • Nieves Capote
    • 2
  • Carlos J. López-Herrera
    • 1
    Email author
  1. 1.Instituto de Agricultura Sostenible, CSICCórdobaSpain
  2. 2.IFAPA Centro Las TorresAlcalá del RíoSpain

Personalised recommendations