, Volume 35, Issue 2, pp 201–214 | Cite as

Temporal patterns of airborne Phytophthora spp. in a woody plant nursery area detected using real-time PCR

  • Duccio MiglioriniEmail author
  • Luisa Ghelardini
  • Nicola Luchi
  • Paolo Capretti
  • Marzia Onorari
  • Alberto Santini
Original Paper


In this study, spore trap monitoring was applied to provide a proof of concept for the use of qPCR to detect Phytophthora in aerial samples and provide valuable information for epidemiological studies in nurseries. Two qPCR TaqMan assays were developed to detect pathogen DNA: the first used a generic probe to detect Phytophthora spp., and the second was based on a specific probe for detecting P. ramorum and P. lateralis. All samples tested positive for the genus Phytophthora, although P. ramorum and P. lateralis were not detected. In late spring and in autumn, two main peaks of Phytophthora sporulation were observed. Peaks were preceded by rainfall, high relative humidity, and mild temperature. From mid-May to the end of August, Phytophthora DNA detected in the air increased with relative humidity, while it decreased with increasing mean temperature. There was also a positive correlation between Phytophthora DNA detected and rainfall in the same period. No significant correlations between Phytophthora DNA and temperature or rainfall were found from the end of August to December. Our results are in agreement with those obtained with classical diagnostic methods based on microscopy, but the approach used here enabled rapid detection and relative quantification of the target organisms, thus assisting in the implementation of disease management strategies.


Airborne sporangia TaqMan® MGB probe Quantitative PCR Aerial Phytophthora Disease management 



This work was supported by the European Union Seventh Framework Programme FP7 2007–2013 (KBBE 2009-3) under grant agreement 245268 Increasing Sustainability of European Forests: Modelling for Security Against Invasive Pests and Pathogens under Climate Change (ISEFOR). We are grateful to Dr. Thomas Jung (Mendel University, Brno, Czech Republic), Dr. Cecile Robin (INRA, Bordeaux, France), Dr. S. Leonhard and Dr. J. Schumacher (Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, Germany) and Dr. N. Silouti (NAGREF-Institute of Mediterranean Forest Ecosystems Terma Alkmanos, Athens, Greece) for providing part of the isolates and DNA samples of Phytophthora species used in this work. We wormly thank Dr. Trudy Paap for critical revision of the manuscript and Eglish language editing.


  1. Almquist, C., & Wallenhammar, A. C. (2015). Monitoring of plant and airborne inoculum of Sclerotinia sclerotiorum in spring oilseed rape using real-time PCR. Plant Pathology, 64(1), 109–118. Scholar
  2. Aylor, D. E., Schmale, D. G., Shields, E. J., Newcomb, M., & Nappo, C. J. (2011). Tracking the potato late blight pathogen in the atmosphere using unmanned aerial vehicles and Lagrangian modeling. Agricultural and Forest Meteorology, 151(2), 251–260. Scholar
  3. Becktell, M. C., Daughtrey, M. L., & Fry, W. E. (2005). Temperature and leaf wetness requirements for pathogen establishment, incubation period, and sporulation of Phytophthora infestans on Petunia × hybrida. Plant Disease, 89, 975–979. Scholar
  4. Botella, L., Dvořák, M., Bačová, A., Pepori, A. L., Ghelardini, L., & Luchi, N. (2017). Detection and quantification of the air inoculum of Caliciopsis pinea in a plantation of Pinus radiata in Italy. In Invasive Forest Pathogens & Implications for Biology & Policy, IUFRO Working Party 7.02.02 May 7‐11, 2017 Niagara Falls, Ontario (USA). Book of Abstracts, 27 (p. 27).
  5. Brasier, C., & Webber, J. (2010). Plant pathology: Sudden larch death. Nature, 466(7308), 824–825.CrossRefGoogle Scholar
  6. Brown, J. K. M., & Hovmøller, M. S. (2002). Aerial dispersal of fungi on the global and continental scales and its consequences for plant disease. Science, 297(July), 537–541. Scholar
  7. Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., et al. (2009). The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55(4), 611–622. Scholar
  8. Calderon, C., Ward, E., Freeman, J., Foster, S. J., & McCartney, H. A. (2002). Detection of airborne inoculum of Leptosphaeria maculans and Pyrenopeziza brassicae in oilseed rape crops by polymerase chain reaction (PCR) assays. Plant Pathology, 51(3), 303–310. Scholar
  9. Carisse, O., Tremblay, D. M., Lévesque, C. A., Gindro, K., Ward, P., & Houde, A. (2009). Development of a TaqMan Real-Time PCR assay for quantification of airborne conidia of Botrytis squamosa and management of Botrytis leaf blight of onion. Phytopathology, 99(11), 1273–1280. Scholar
  10. Chandelier, A., André, F., & Laurent, F. (2010). Detection of Chalara fraxinea in common ash (Fraxinus excelsior) using real time PCR. Forest Pathology, 40(2), 87–95. Scholar
  11. Crosier, W. (1934). Studies in the biology of Phytophthora infestans (Mont) de Bary. Google Scholar
  12. Davidson, J. M., Wickland, A. C., Patterson, H. A., Falk, K. R., & Rizzo, D. M. (2005). Transmission of Phytophthora ramorum in mixed-evergreen forest in California. Phytopathology, 95(5), 587–596. Scholar
  13. Denman, S., Kirk, S. A., Moralejo, E., & Webber, J. F. (2009). Phytophthora ramorum and Phytophthora kernoviae on naturally infected asymptomatic foliage. EPPO Bulletin, 39(1), 105–111.CrossRefGoogle Scholar
  14. Dick, M., & Parke, J. L. (2012). Phythophthora kernoviae. Forest Phytophthoras. Scholar
  15. Duvivier, M., Dedeurwaerder, G., De Proft, M., Moreau, J. M., & Legrève, A. (2013). Real-time PCR quantification and spatio-temporal distribution of airborne inoculum of Mycosphaerella graminicola in Belgium. European Journal of Plant Pathology, 137(2), 325–341. Scholar
  16. Duvivier, M., Dedeurwaerder, G., Marchal, G., Renard, M. E., Van Hese, V., Moreau, J. M., et al. (2010). Distribution of airborne Mycosphaerella graminicola inoculum at the field scale. Communications in Agricultural and Applied Biological Sciences, 75, 635–639.Google Scholar
  17. Dvořák, M., Janoš, P., Botella, L., Rotková, G., & Zas, R. (2017). Spore dispersal pattern of Fusarium circinatum on an infected Monterey pine forest in north‐western Spain. In Invasive Forest Pathogens & Implications for Biology & Policy, IUFRO Working Party 7.02.02 May 7‐11, 2017 Niagara Falls, Ontario (USA) (p. 23).
  18. Dvorak, M., Rotkova, G., & Botella, L. (2015). Detection of airborne inoculum of Hymenoscyphus fraxineus and H. albidus during seasonal fluctuations associated with absence of apothecia. Forests, 7(1), 1–13. Scholar
  19. Eppo Global Database. (2011). New additions to the EPPO Lists. EPPO Reporting Service no. 09 - 2011. Num. article: 2011/187.
  20. Eppo Global Database. (2013). New additions to the EPPO A1 and A2 Lists. EPPO Reporting Service no. 09 - 2013. Num. article: 2013/197.
  21. Erwin, D. C., & Ribeiro, O. K. (1996). Phytophthora: Diseases worldwide. St. Paul, MN: APS Press.Google Scholar
  22. EU Commition Decision. (2002). Commission Decision (2002/757/EC) on provisional emergency phytosanitary measures to prevent the introduction into and the spread within the Community of Phytophthora ramorum Werres, De Cock & Man in ‘t Veld sp. nov. Official Journal of the European Communities, L.252, 37–39.Google Scholar
  23. EU Commition Decision. (2004). Commission Decision (2004/426/EC) amending Decision 2002/757/EC on provisional emergency phytosanitary measures to prevent the introduction into and the spread within the Community of Phytophthora ramorum Werres, De Cock & Man in ‘t Veld sp. nov. Official Journal of the European Union, L.154, 1–3.Google Scholar
  24. EU Commition Decision. (2007). Commission Decision (2007/201/EC) amending Decision 2002/757/EC on provisional emergency phytosanitary measures to prevent the introduction into and the spread within the Community of Phytophthora ramorum Werres, De Cock & Man in ‘t Veld sp. nov. Official Journal of the European Union, L.90, 83–85.Google Scholar
  25. Fall, M. L., Tremblay, D. M., Gobeil-Richard, M., Couillard, J., Rocheleau, H., Van Der Heyden, H., et al. (2015a). Infection efficiency of four Phytophthora infestans clonal lineages and DNA-based quantification of sporangia. PLoS ONE, 10(8), 1–18. Scholar
  26. Fall, M. L., Van der Heyden, H., Brodeur, L., Leclerc, Y., Moreau, G., & Carisse, O. (2015b). Spatiotemporal variation in airborne sporangia of Phytophthora infestans: Characterization and initiatives towards improving potato late blight risk estimation. Plant Pathology, 64(1), 178–190. Scholar
  27. Fang, Y., & Ramasamy, R. P. (2015). Current and prospective methods for plant disease detection. Biosensors, 5(3), 537–561. Scholar
  28. Fountaine, J. M., Shaw, M. W., Ward, E., & Fraaije, B. A. (2010). The role of seeds and airborne inoculum in the initiation of leaf blotch (Rhynchosporium secalis) epidemics in winter barley. Plant Pathology, 59(2), 330–337. Scholar
  29. Garbelotto, M., Smith, T., & Schweigkofler, W. (2008). Variation in rates of spore deposition of Fusarium circinatum, the causal agent of pine pitch canker, over a 12-month-period at two locations in Northern California. Phytopathology, 98(1), 137–143. Scholar
  30. Gerlach, W. W. P., Hoitink, H. A. J., & Schmitthenner, A. F. (1976). Phytophthora citrophthora on Pieris japonica: Infection, sporulation, and disemination. Phytopathology, 66, 302–308.CrossRefGoogle Scholar
  31. Ginetti, B., Carmignani, S., Ragazzi, A., Werres, S., & Moricca, S. (2014). Foliar blight and shoot dieback caused by Phytophthora ramorum on Viburnum tinus in the Pistoia area, Tuscany, central Italy. Plant Disease, 98(3), 423. Scholar
  32. Goth, R. W., & Wester, R. E. (1963). Culture of Phytophthora phaseoli on living and sterilized media. Phytopathology, 53(2), 233.Google Scholar
  33. Green, S., Brasier, C. M., Schlenzig, A., Mccracken, A., Macaskill, G. A., Wilson, M., et al. (2013). The destructive invasive pathogen Phytophthora lateralis found on Chamaecyparis lawsoniana across the UK. Forest Pathology, 43(1), 19–28. Scholar
  34. Green, S., Elliot, M., Armstrong, A., & Hendry, S. J. (2014). Phytophthora austrocedrae emerges as a serious threat to juniper (Juniperus communis) in Britain. Plant Pathology, 64(2), 456–466. Scholar
  35. Green, S., Hendry, S. J., Macaskill, G. A., Laue, B. E., & Steele, H. (2012). Dieback and mortality of Juniperus communis in Britain associated with Phytophthora austrocedrae. New Disease Reports, 26(2), 5197. Scholar
  36. Gregory, P. H., & Hirst, J. M. (1957). The summer air-spora at Rothamsted in 1952. Journal of General Microbiology, 17(1957), 135–152. Scholar
  37. Greslebin, A. G., Hansen, E. M., & Sutton, W. (2007). Phytophthora austrocedrae sp. nov., a new species associated with Austrocedrus chilensis mortality in Patagonia (Argentina). Mycological Research, 111(3), 308–316. Scholar
  38. Grove, G. G., Madden, L. V., & Ellis, M. A. (1985). Influence of temperature and wetness duration on sporulation of Phytophthora cactorum on infected strawberry fruit. Phytopathology, 75(6), 700–703.CrossRefGoogle Scholar
  39. Gullino, C., Garofalo, M. C., Moretti, F., Gianetti, G., & Mainenti, E. (2003). Rinvenimento su rododendro di Phytophthora ramorum. L’Informatore Agrario, 19, 87–89.Google Scholar
  40. Hansen, E. M. (2011). Phytophthora lateralis. Forest Phytophthoras. Scholar
  41. Hansen, E. M. (2012). Phytophthora pinifolia. Forest Phytophthoras. Scholar
  42. Heard, S., & West, J. S. (2014). New developments in identification and quantification of airborne inoculum. In M. L. Gullino & P. J. M. Bonants (Eds.), Detection and diagnostics of plant pathogens, plant pathology in the 21st century (5th ed., pp. 3–18). Dordrecht: Springer. Scholar
  43. Jung, T., Orlikowski, L., Henricot, B., Abad-Campos, P., Aday, A. G., Aguín Casal, O., et al. (2016). Widespread Phytophthora infestations in European nurseries put forest, semi-natural and horticultural ecosystems at high risk of Phytophthora diseases. Forest Pathology, 46(2), 134–163. Scholar
  44. Klosterman, S. J., Anchieta, A., McRoberts, N., Koike, S. T., Subbarao, K. V., Voglmayr, H., et al. (2014). Coupling spore traps and quantitative PCR assays for detection of the downy mildew pathogens of spinach (Peronospora effusa) and beet (P. schachtii). Phytopathology, 104(12), 1349–1359. Scholar
  45. Kroon, L. P. N. M., Brouwer, H., de Cock, A. W. A. M., & Govers, F. (2012). The genus Phytophthora Anno 2012. Phytopathology, 102(4), 348–364. Scholar
  46. Lacey, J. (1996). Spore dispersal—Its role in ecology and disease: The British contribution to fungal aerobiology. Mycological Research, 100(6), 641–660. Scholar
  47. Luchi, N., Ghelardini, L., Belbahri, L., Quartier, M., & Santinia, A. (2013). Rapid detection of Ceratocystis platani inoculum by quantitative real-time PCR assay. Applied and Environmental Microbiology. Scholar
  48. Manzano, J. M. M., Molina, R. T., Rodríguez, S. F., Barroso, P. D., Palacios, I. S., & Garijo, Á. G. (2015). Airborne propagules of Phytophthora and related taxa in SW Spain including a predictive model. European Journal of Plant Pathology, 143(3), 473–483. Scholar
  49. Martinelli, F., Scalenghe, R., Davino, S., Panno, S., Scuderi, G., Ruisi, P., et al. (2015). Advanced methods of plant disease detection. A review. Agronomy for Sustainable Development, 35(1), 1–25. Scholar
  50. Meitz-Hopkins, J. C., von Diest, S. G., Koopman, T. A., Bahramisharif, A., & Lennox, C. L. (2014). A method to monitor airborne Venturia inaequalis ascospores using volumetric spore traps and quantitative PCR. European Journal of Plant Pathology, 140(3), 527–541. Scholar
  51. Migliorini, D., Ghelardini, L., Tondini, E., Luchi, N., & Santini, A. (2015). The potential of symptomless potted plants for carrying invasive soilborne plant pathogens. Diversity and Distributions, 21(10), 1218–1229. Scholar
  52. Parke, J. L., & Rizzo, D. M. (2011). Phytophthora ramorum. Forest Phytophthoras. Scholar
  53. Party, I. W. (2017). Conference Program Invasive Forest Pathogens & Implications for Biology & Policy Niagara Falls, Ontario.Google Scholar
  54. Reeser, P., Sutton, W., & Hansen, E. (2011). Phytophthora species in tanoak trees, canopy-drip, soil, and streams in the sudden oak death epidemic area of south-western Oregon, USA. New Zealand Journal of Forestry Science, 41(SUPPL), 65–73.Google Scholar
  55. Reeser, P., Sutton, W., & Hansen, E. (2013). Phytophthora pluvialis, a new species from mixed tanoak-Douglas-fir forests of western Oregon, U.S.A. North American Fungi, 8(7), 1–8. Scholar
  56. Ribeiro, O. K. (1983). Phytophthora: its Biology, Taxonomy, Ecology and Pathology. (D. C. Erwin, S. Bartnicki-Garcia, & P. H. Tsao, Eds.). St Paul, Minnesota: APS Press.Google Scholar
  57. Robin, C., Piou, D., Feau, N., Douzon, G., Schenck, N., & Hansen, E. M. (2011). Root and aerial infections of Chamaecyparis lawsoniana by Phytophthora lateralis: A new threat for European countries. Forest Pathology, 41(5), 417–424. Scholar
  58. Rogers, S. L., Atkins, S. D., & West, J. S. (2009). Detection and quantification of airborne inoculum of Sclerotinia sclerotiorum using quantitative PCR. Plant Pathology, 58(2), 324–331. Scholar
  59. Rooney-Latham, S., Honeycutt, E., Ochoa, J., Grünwald, N. J., & Blomquist, C. L. (2013). First Report of camphor tree (Cinnamomum camphora) as a host of Phytophthora ramorum. Plant Disease, 97(10), 1377. Scholar
  60. Sails, A., & Tang, Y. W. (2015). Methods in microbiology. Cambridge: Academic Press. Scholar
  61. Santini, A., Barzanti, G. P., & Capretti, P. (2001). A new Phytophthora root disease of alder in Italy. Plant Disease, 85(5), 560. Scholar
  62. Santini, A., Ghelardini, L., De Pace, C., Desprez-Loustau, M. L., Capretti, P., Chandelier, A., et al. (2013). Biogeographical patterns and determinants of invasion by forest pathogens in Europe. New Phytologist, 197(1), 238–250. Scholar
  63. Schweigkofler, W., & Garbelotto, M. (2004). Detection and quantification of airborne conidia of Fusarium circinatum, the causal agent of pine pitch canker, from two California sites by using a real-time PCR approach combined with a simple spore trapping method. Applied and Environmental Microbiology, 70(6), 3512–3520. Scholar
  64. Team RC. (2015). R: A language and environment for statistical computing. R Foundation for Statistical Computing.Google Scholar
  65. Techy, L., Schmale, D. G., & Woolsey, C. A. (2010). Coordinated aerobiological sampling of a plant pathogen in the lower atmosphere using two autonomous unmanned aerial vehicles. J. Field Robotics, 27(3), 335–343. Scholar
  66. Timmer, L. W., Zitko, S. E., Gottwald, T. R., & Graham, J. H. (2000). Phytophthora brown rot of Citrus: temperature and moisture effects on infection, sporangium production, and dispersal. Plant Disease, 84(2), 157–163. Scholar
  67. Tooley, P. W., & Browning, M. (2015). Temperature effects on the onset of sporulation by Phytophthora ramorum on Rhododendron “Cunningham’s White”. Journal of Phytopathology, 163(11–12), 908–914. Scholar
  68. Trujillo, E. E. (1965). The effects of humidity and temperature on Phytophthora blight of taro. Phytopathology, 55, 183–188.Google Scholar
  69. Università degli Studi di Firenze. (2016). Indagine sul settore vivaistico ornamentale in Toscana - 2016. Accessed 1 August 2017
  70. Warnes, G. R., Bolker, B., Bonebakker, L., Robert Gentleman, Liaw, W. H. A., Lumley, T., et al. (2016). gplots: various R programming tools for plotting data. R package version 3.0.1. The Comprehensive R Archive Network.Google Scholar
  71. Webber, J. F., Mullett, M., & Brasier, C. M. (2010). Dieback and mortality of plantation Japanese larch (Larix kaempferi) associated with infection by Phytophthora ramorum. New Disease Reports, 22, 19. Scholar
  72. Webber, J. F., Vettraino, A. M., Chang, T. T., Bellgard, S. E., Brasier, C. M., & Vannini, A. (2012). Isolation of Phytophthora lateralis from Chamaecyparis foliage in Taiwan. Forest Pathology, 42(2), 136–143. Scholar
  73. West, J. S., Atkins, S. D., Emberlin, J., & Fitt, B. D. L. (2008). PCR to predict risk of airborne disease. Trends in Microbiology, 16(8), 380–387. Scholar

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© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Istituto per la Protezione Sostenibile delle Piante – C.N.R.Sesto fiorentinoItaly
  2. 2.Dipartimento di Scienze delle Produzioni Agroalimentari e dell’Ambiente DiSPAAUniversità di FirenzeFlorenceItaly
  3. 3.Agenzia Regionale per la Protezione Ambientale della Toscana. A.F.R. AerobiologiaPistoiaItaly

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