Variation in the nrDNA ITS sequences of some powdery mildew species: do routine molecular identification procedures hide valuable information?

  • Gábor M. Kovács
  • Tünde Jankovics
  • Levente Kiss
Article

Abstract

During the past years, nrDNA ITS sequences have supported the identification of many powdery mildew fungi because comprehensive analyses showed that differences in these sequences have always correlated with the delimitation of different species and formae speciales of the Erysiphales. Published data, obtained using direct sequencing of the PCR products, suggested that even one to five nucleotide differences in the ITS sequences delimit different, albeit closely related, species, and/or indicate differences in host range patterns. Here we show that such differences in the ITS sequences can be detected even in a single sample of a powdery mildew fungus. We sequenced the ITS region in 17 samples, representing six powdery mildew species, both directly and after cloning the PCR products. Among these, samples of O. longipes exhibited two or three, samples of O. neolycopersici three or four, those of an Oidium sp. from Chelidonium majus up to seven, and a sample of another Oidium sp. from Passiflora caerulea two different ITS types determined after cloning. No ITS nucleotide polymorphisms were found in samples of O. lycopersici and Erysiphe aquilegiae. This suggests that some powdery mildew taxa are more variable at the ITS level than others. Thus, although the ITS sequences determined by direct sequencing represent robust data useful in delimitation and phylogenetic analysis of distinct species of the Erysiphales, these need to be used with precaution, and preferably determined after cloning, especially when dealing with closely related taxa at species and sub-species levels. With this method a hitherto undetected genetic diversity of powdery mildews can be revealed.

Keywords

Erysiphales GenBank ITS polymorphism paralogue sequences 

References

  1. Alaei, H., De Backer, M., Nuytinck, J., Maes, M., Höfte, M., & Heungens, K. (2009). Phylogenetic relationships of Puccinia horiana and other rust pathogens of Chrysanthemum x morifolium based on rDNA ITS sequence analysis. Mycological Research, 113, 668–683.PubMedCrossRefGoogle Scholar
  2. Beilharz, V., Cunnington, J. H., & Pascoe, I. G. (2010). Neoerysiphe kerribeeensis sp. nov. (Ascomycota: Erysiphales), a new species of Neoerysiphe on native and introduced species of Senecio (Asteraceae) in Australia. Fungal Biology, 114, 340–344.PubMedCrossRefGoogle Scholar
  3. Brewer, M. T., & Milgroom, M. G. (2010). Phylogeography and population structure of the grape powdery mildew fungus, Erysiphe necator, from diverse Vitis species. BMC Evolutionary Biology, 10, 268. doi:10.1186/1471-2148-10-268.PubMedCrossRefGoogle Scholar
  4. Bridge, P. D., Roberts, P. J., Spooner, B. M., & Panchal, G. (2003). On the unreliability of published DNA sequences. The New Phytologist, 160, 43–48.CrossRefGoogle Scholar
  5. Cook, R. T. A., Henricot, B., Henrici, A., & Beales, P. (2006). Morphological and phylogenetic comparisons amongst powdery mildews on Catalpa in the UK. Mycological Research, 110, 672–685.PubMedCrossRefGoogle Scholar
  6. Cunnington, J. H., Takamatsu, S., Lawrie, A. C., & Pascoe, I. G. (2003). Molecular identification of anamorphic powdery mildews (Erysiphales). Australasian Plant Pathology, 32, 421–428.CrossRefGoogle Scholar
  7. Cunnington, J. H., Lawrie, A. C., & Pascoe, I. G. (2005). Genetic variation within Podosphaera tridactyla reveals a paraphyletic species complex with biological specialization towards specific Prunus subgenera. Mycological Research, 119, 357–362.CrossRefGoogle Scholar
  8. Eberhardt, U. (2010). A constructive step towards selecting a DNA barcode for fungi. The New Phytologist, 187, 265–268.PubMedCrossRefGoogle Scholar
  9. Heluta, V., Takamatsu, S., Voytyuk, S., & Shiroya, Y. (2009). Erysiphe kenjiana (Erysiphales), a new invasive fungus in Europe. Mycological Progress, 8, 367–375.CrossRefGoogle Scholar
  10. Hirose, S., Tanda, S., Kiss, L., Grigaliunaite, B., Havrylenko, M., & Takamatsu, S. (2005). Molecular phylogeny and evolution of the maple powdery mildew (Sawadaea, Erysiphaceae) inferred from nuclear rDNA sequences. Mycological Research, 109, 912–922.PubMedCrossRefGoogle Scholar
  11. Inuma, T., Khodaparast, S. A., & Takamatsu, S. (2007). Multilocus phylogenetic analyses within Blumeria graminis, a powdery mildew fungus of cereals. Molecular Phylogenetics and Evolution, 44, 741–751.PubMedCrossRefGoogle Scholar
  12. Ito, M., & Takamatsu, S. (2010). Molecular phylogeny and evolution of subsection Magnicellulatae (Erysiphaceae: Podosphaera) with special reference to host plants. Mycoscience, 51, 34–43.CrossRefGoogle Scholar
  13. Jankovics, T., Bai, Y., Kovács, G. M., Bardin, M., Nicot, P. C., Toyoda, H., et al. (2008). Oidium neolycopersici: intra-specific variability inferred from AFLP analysis and relationship with closely related powdery mildew fungi infecting various plant species. Phytopathology, 98, 529–540.PubMedCrossRefGoogle Scholar
  14. Kang, S., Mansfield, M. A., Park, B., Geiser, D. M., Ivors, K. L., Coffey, M. D., et al. (2010). The promise and pitfalls of sequence-based identification of plant pathogenic fungi and oomycetes. Phytopathology, 100, 732–737.PubMedCrossRefGoogle Scholar
  15. Kiss, L., Takamatsu, S., & Cunnington, J. H. (2005). Molecular identification of Oidium neolycopersici as the casual agent of the recent tomato powdery mildew epidemics in North America. Plant Disease, 89, 491–496.CrossRefGoogle Scholar
  16. Kiss, L., Khosla, K., Jankovics, T., Niinomi, S., Braun, U., & Takamatsu, S. (2006). A morphologically ill-founded powdery mildew species, Pleochaeta indica, is recognized as a phylogenetic species based on the analysis of the nuclear ribosomal DNA sequences. Mycological Research, 110, 1301–1308.PubMedCrossRefGoogle Scholar
  17. Kiss, L., Jankovics, T., Kovács, G. M., & Daughtrey, M. L. (2008). Oidium longipes, a new powdery mildew fungus on petunia in the USA: a potential threat to ornamental and vegetable solanaceous crops. Plant Disease, 92, 818–825.CrossRefGoogle Scholar
  18. Kovács, G. M., Balázs, T., & Pénzes, Z. (2007). Molecular study of arbuscular mycorrhizal fungi colonizing the sporophyte of the eusporangiate rattlesnake fern (Botrychium virginianum, Ophioglossaceae). Mycorrhiza, 17, 597–605.PubMedCrossRefGoogle Scholar
  19. Kovács, G. M., Trappe, J. M., Alsheikh, A. M., Bóka, K., & Elliott, T. F. (2008). Imaia, a new truffle genus to accommodate Terfezia gigantea. Mycologia, 100, 930–939.PubMedCrossRefGoogle Scholar
  20. Kovács, G. M., Balázs, T. K., Calonge F. D. & Martín, M. P. (2011). The diversity of Terfezia desert truffles: new species and a highly variable species complex with intrasporocarpic nrDNA ITS heterogeneity. Mycologia (in press)Google Scholar
  21. Leus, L., Dewitte, A., Van Huylenbroeck, J., Vanhoutte, N., Van Bockstaele, E., & Hofte, M. (2006). Podosphaera pannosa (syn. Sphaerotheca pannosa) on Rosa and Prunus spp.: characterization of pathotypes by differential plant reactions and ITS sequences. Journal of Phytopathology, 154, 23–28.CrossRefGoogle Scholar
  22. Matsuda, S., & Takamatsu, S. (2003). Evolution of host-parasite relationship of Golovinomyces (Ascomycete: Erysiphaceae) inferred from nuclear rDNA sequences. Molecular Phylogenetics and Evolution, 27, 314–327.PubMedCrossRefGoogle Scholar
  23. Nilsson, R. H., Kristiansson, E., Ryberg, M., Hallenberg, N., & Larsson, K. H. (2008). Intraspecific ITS variability in the Kingdom Fungi as expressed in the international sequence databases and its implications for molecular species identification. Evolutionary Bioinformatics, 4, 193–201.Google Scholar
  24. O’Donnell, K., & Cigelnik, E. (1997). Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Molecular Phylogenetics and Evolution, 7, 103–116.PubMedCrossRefGoogle Scholar
  25. Seko, Y., Bolay, A., Kiss, L., Heluta, V., Grigaliunaite, B., & Takamatsu, S. (2008). Molecular evidence in support of recent migration of a powdery mildew fungus on Syringa spp. into Europe from East Asia. Plant Pathology, 57, 243–250.CrossRefGoogle Scholar
  26. Shiroya, Y., & Takamatsu, S. (2009). Erysiphe corylopsidis sp. nov., a new powdery mildew fungus found on Corylopsis spicata and C. pauciflora. Mycoscience, 50, 409–414.CrossRefGoogle Scholar
  27. Simon, U. K., & Weiss, M. (2008). Intragenomic variation of fungal ribosomal genes is higher than previously thought. Molecular Biology and Evolution, 25, 2251–2254.PubMedCrossRefGoogle Scholar
  28. Smith, M. E., Douhan, G. W., & Rizzo, D. M. (2007). Intra-specific and intra-sporocarp ITS variation of ectomycorrhizal fungi as assessed by rDNA sequencing of sporocarps and pooled ectomycorrhizal roots from a Quercus woodland. Mycorrhiza, 18, 15–22.PubMedCrossRefGoogle Scholar
  29. Szentiványi, O., Kiss, L., Russell, J. C., Kovács, G. M., Varga, K., Jankovics, T., et al. (2005). Ampelomyces mycoparasites from apple powdery mildew identified as a distinct group based on single-stranded conformation polymorphism analysis of the rDNA ITS region. Mycological Research, 109, 429–438.PubMedCrossRefGoogle Scholar
  30. Rossman, A. (2007). Report of the planning workshop for all fungi DNA barcoding. Inoculum, 58(6), 1–5.Google Scholar
  31. Takamatsu, S., Braun, U., Limkaisang, S., Kom-una, S., Sato, Y., & Cunnington, J. H. (2007). Phylogeny and taxonomy of the oak powdery mildew Erysiphe alphitoides sensu lato. Mycological Research, 111, 809–826.PubMedCrossRefGoogle Scholar
  32. Takamatsu, S., Inagaki, M., Niinomi, S., Khodaparast, S. A., Shin, H.-D., Grigaliunaite, B., et al. (2008). Comprehensive molecular phylogenetic analysis and evolution of the genus Phyllactinia (Ascomycota: Erysiphales) and its allied genera. Mycological Research, 112, 299–315.PubMedCrossRefGoogle Scholar
  33. Takamatsu, S., Heluta, V., Havrylenko, M., & Divarangkoon, R. (2009). Four powdery mildew species with catenate conidia infect Galium: molecular and morphological evidence. Mycological Research, 113, 117–129.PubMedCrossRefGoogle Scholar
  34. Vági, P., Kovács, G. M., & Kiss, L. (2007). Host range expansion in a powdery mildew fungus (Golovinomyces sp.) infecting Arabidopsis thaliana: Torenia fournieri as a new host. European Journal of Plant Pathology, 117, 89–93.CrossRefGoogle Scholar
  35. Wyand, R. A., & Brown, J. K. M. (2003). Genetic and forma specialis diversity in Blumeria graminis of cereals and its implications for host pathogen co-evolution. Molecular Plant Pathology, 4, 187–198.PubMedCrossRefGoogle Scholar

Copyright information

© KNPV 2011

Authors and Affiliations

  • Gábor M. Kovács
    • 1
    • 2
  • Tünde Jankovics
    • 1
  • Levente Kiss
    • 1
  1. 1.Plant Protection Institute of the Hungarian Academy of SciencesBudapestHungary
  2. 2.Department of Plant AnatomyEötvös Loránd UniversityBudapestHungary

Personalised recommendations