Skip to main content

Advertisement

SpringerLink
Log in

Characterization and development of qPCR for early detection and quantification of Pseudocercospora macadamiae at different stages of infection process

  • Published:
European Journal of Plant Pathology Aims and scope Submit manuscript

Abstract

Ability to detect Pseudocercospora macadamiae infection in macadamia husk at least four months before symptoms become visible will aid the development of disease control measures. This study examined the distinctness of P. macadamiae within the phylogenetic lineages of the genus Pseudocercospora. In addition, we developed two quantitative PCR (qPCR) assays, as rapid diagnostic tools, for early detection and quantification of P. macadamiae in planta. Phylogenetic analysis of concatenated sequences of four gene loci (large subunits, internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α) and actin of 47 P. macadamiae isolates showed that P. macadamiae is a distinct species in the genus Pseudocercospora. P. macadamiae isolates were partitioned into subunits in the cluster but the grouping of the isolates was regardless of location. Nucleotide diversity (0.02) and the coefficient of genetic differentiation (0.07) were low in the P. macadamiae population. Two qPCR primer sets, based on ITS (PMI) and TEF-1α (PME) were designed that consistently amplified P. macadamiae in fungal cultures (Ct = 16.93 ± 0.11 and Ct = 21.20 ± 0.11, respectively) and in planta (Ct = 32.36 ± 0.28 and Ct = 38.07 ± 1.20, respectively). The PMI primers also detected species in the genus Pseudocercospora, while PME was more specific and robust for quantification of P. macadamiae. Both primer sets detected P. macadamiae in asymptomatic tissue samples and strongly differentiated various stages of disease progression, which revealed approximately 10-fold increase in fungal biomass between each consecutive stage of symptom development.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Akinsanmi, O. A., Miles, A. K., & Drenth, A. (2007). Timing of fungicide application for control of husk spot caused by Pseudocercospora macadamiae in macadamia. Plant Disease, 91, 1675–1681.

    Article  CAS  Google Scholar 

  • Akinsanmi, O. A., Miles, A. K., & Drenth, A. (2008). Alternative fungicides for controlling husk spot caused by Pseudocercospora macadamiae in macadamia. Australasian Plant Pathology, 37, 141–147.

    Article  CAS  Google Scholar 

  • Akinsanmi, O. A., Topp, B., & Drenth, A. (2012). Pericarps retained in the tree canopy and stomatal abundance are components of resistance to husk spot caused by Pseudocercospora macadamiae in macadamia. Euphytica, 185, 313–323.

    Article  CAS  Google Scholar 

  • Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403–410.

    Article  CAS  PubMed  Google Scholar 

  • Beilharz, V., Mayers, P. E., & Pascoe, I. G. (2003). Pseudocercospora macadamiae sp. nov., the cause of husk spot of macadamia. Australas. Plant Pathology, 32, 279–282.

    Google Scholar 

  • Brisson, M., Hall, S., Hamby, R. K., Park, R., & Srere, H. K. (Eds.) (2004). Optimisation of single and multiplex real-time PCR. USA: International University Line.

    Google Scholar 

  • Capote, N., Pastrana, A.M., 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, pp. 151–202.

  • Carbone, I., & Kohn, L. M. (1999). A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia, 91, 553–556.

    Article  CAS  Google Scholar 

  • Crous, P. W., Braun, U., Hunter, G. C., Wingfield, M. J., Verkley, G. J. M., Shin, H.-D., et al. (2012). Phylogenetic lineages in Pseudocercospora. Studies in Mycology, 75(1), 37–114.

  • Deighton, F. C. (1976). Studies on Cercospora and allied genera. VI. Pseudocercospora Speg., Pantospora Cif. And Cercoseptoria Petr. Mycological Papers, 140, 1–168.

    Google Scholar 

  • Drenth, A., Akinsanmi, O. A., & Miles, A. K. (2009). Macadamia diseases in Australia. Southern African Macadamia Growers’ Association Yearbook, 17, 48–52.

    Google Scholar 

  • Francis, W. D. (1928). The anatomy of the Australia bush nut (Macadamia ternifolia). Proc. R. Soc. Queensl., 39, 43–53.

    Google Scholar 

  • Hartl, D. L., & Clark, A. G. (1997). Principles of population genetics. Sunderland, MA: Sinauer Associates, Inc..

    Google Scholar 

  • Hasegawa, E., Ota, Y., Hattori, T., & Kikuchi, T. (2010). Sequence-based identification of Japanese Armillaria species using the elongation factor-1 alpha gene. Mycologia, 102, 898–910.

    Article  CAS  PubMed  Google Scholar 

  • Kristensen, R., Torp, M., Kosiak, B., & Holst-Jensen, A. (2005). Phylogeny and toxigenic potential is correlated in Fusarium species as revealed by partial translation elongation factor-1 alpha gene sequences. Mycological Research, 109, 173–186.

    Article  CAS  PubMed  Google Scholar 

  • Leina, M. J., Tan, T. K., & Wong, S. M. (1996). Resistance of Hibiscus esculentus L. And Vigna sinensis (L.) Endl. To Pseudocercospora and plant peroxidase activity in relation to infection. The Annals of Applied Biology, 129, 197–206.

    Article  CAS  Google Scholar 

  • Librado, P., & Rozas, J. (2009). DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25, 1451–1452.

    Article  CAS  PubMed  Google Scholar 

  • Mayers, P. E., & Hutton, D. G. (1987). Husk spot of macadamia in Queensland and its control. In T. Trochoulias & I. Skinner (Eds.), Proceedings of the second Australian macadamia research workshop (pp. 146–151). Bangalow: Australian Macadamia Society.

    Google Scholar 

  • McDermott, J. M., & McDonald, B. A. (1993). Gene flow in plant pathosystems. Annual Review of Phytopathology, 31, 353–373.

    Article  Google Scholar 

  • Miles, A. K. (2011). Husk spot disease of macadamia. Phd Thesis. Brisbane: University of Queenland.

    Google Scholar 

  • Miles, A. K., Akinsanmi, O. A., Sutherland, P. W., Aitken, E. A. B., & Drenth, A. (2009). Infection, colonisation and sporulation by Pseudocercospora macadamiae on macadamia fruit. Australasian Plant Pathology, 38, 36–43.

    Article  Google Scholar 

  • Miles, A. K., Akinsanmi, O. A., Aitken, E. A. B., & Drenth, A. (2010a). Timing of infection of macadamia fruit by Pseudocercospora macadamiae and climatic effects on growth and spore germination. Australasian Plant Pathology, 39, 453–462.

    Article  Google Scholar 

  • Miles, A. K., Akinsanmi, O. A., Aitken, E. A. B., & Drenth, A. (2010b). Source of Pseudocercospora macadamiae inoculum in macadamia trees and its use for characterising husk spot susceptibility in the field. Crop Protection, 29, 1347–1353.

    Article  Google Scholar 

  • Nei, M. (1973). Analysis of gene diversity in subdivided populations. Proceedings of the National Academy of Sciences of the United States of America, 70, 3321–3323.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Newett, S.D.E., 1983. Observations on cercospora husk spot in macadamia. Proceedings of the first australian macadamia research workshop.

  • Ross-Davis, A. L., Hanna, J. W., Kim, M. S., & Klopfenstein, N. B. (2012). Advances toward DNA-based identification and phylogeny of north American Armillaria species using elongation factor-1 alpha gene. Mycoscience, 53, 161–165.

    Article  CAS  Google Scholar 

  • Seifert, K., & Levesque, C. A. (2004). Phylogeny and molecular diagnosis of mycotoxigenic fungi. European Journal of Plant Pathology, 110, 449–471.

    Article  CAS  Google Scholar 

  • Storey, W. B. (1965). The ternifolia group of Macadamia species. Pacific Science, 15, 507–514.

    Google Scholar 

  • Strohschen, B. (1986). Contributions to the biology of useful plants. IV. Anatomical studies of fruit development and fruit classification of the macadamia nut (Macadamia integrifolia maiden and Betche). Angewandte Botanik, 60, 239–247.

    Google Scholar 

  • Tajima, F. (1989). Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics, 123, 585–595.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA 6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30, 2725–2729.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Vilgalys, R., & Hester, M. (1990). Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology, 172, 4238–4246.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, & T. J. White (Eds.), PCR protocols (pp. 315–322). San Diego: A guide to methods and applications. Academic Press.

    Google Scholar 

  • Wright (1993). Investigations contributing to the knowledge of pathogenicity and genetic variability of the macadamia husk spot pathogen, Pseudocercospora sp. BSc Honours. Brisbane: University of Queensland.

    Google Scholar 

  • Zahn, M., Teuber, F., Bollig, K., & Horst, W. J. (2011). Quantification of Pseudocercospora fuligena in tomato lines carrying introgressions from Solanum habrochaites using a qPCR assay. Plant Disease, 95, 394–400.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

Ong CE acknowledges the scholarship received from the Public Service Department of Malaysia for her Bachelor of Agriculture with Honours degree program at The University of Queensland. The authors are grateful to Ms. Cecilia O′ Dwyer for her technical assistance and the Queensland Plant Pathology Herbarium (BRIP) culture collection for providing the cultures used in this study.

Author information

Authors and Affiliations

  1. Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, Australia

    C. E. Ong, J. Henderson & O. A. Akinsanmi

  2. School of Agriculture and Food Science, The University of Queensland, Qld, Brisbane, 4075, Australia

    C. E. Ong

Authors
  1. C. E. Ong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J. Henderson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. O. A. Akinsanmi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to O. A. Akinsanmi.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ong, C.E., Henderson, J. & Akinsanmi, O.A. Characterization and development of qPCR for early detection and quantification of Pseudocercospora macadamiae at different stages of infection process. Eur J Plant Pathol 147, 85–102 (2017). https://doi.org/10.1007/s10658-016-0982-y

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10658-016-0982-y

Keywords

Search

Navigation