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Development of a quantitative real-time PCR assay using SYBR Green for early detection and quantification of Austropuccinia psidii in Eucalyptus grandis

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Commercial areas containing Eucalyptus plantations have expanded in recent years due to increased demands for pulp, paper and bioenergy. One of the threats that can reduce Eucalyptus production is the eucalyptus rust disease caused by Austropuccinia psidii, a biotrophic fungus that affects a broad range of Myrtaceae. An accurate diagnosis tool for the early detection of rust disease could be useful in breeding programs for selection of resistant plants against rust, in phytosanitary purposes or in rust epidemics studies. The aim of the present work was to develop a SYBR Green-based quantitative real-time PCR (qPCR) assay for the early detection and quantification of A. psidii in Eucalyptus grandis leaves. Three sets of primers based on the A. psidii ribosomal DNA intergenic space region (IGS), beta-tubulin and elongation factor genes were designed and evaluated. The assays using the IGS primer set resulted in the highest detection efficiency, detecting a lower limit of 0.5 pg of A. psidii DNA. Under artificial inoculation in plants, A. psidii was detected immediately after pathogen inoculation until 240 h post-inoculation using qPCR. In field validation of the method, A. psidii was detected using qPCR in naturally infected leaves with or without rust symptoms. This easy and fast method can be used for an efficient detection of A. psidii in E. grandis leaves. The implications of this tool for rust studies are discussed below.

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This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP (Grant 2008/50361-1, 2014/16804-4). We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and FAPESP for the fellowships to A.P.B. (Proc. no.140040/2012-9 and 2013/07596-6), M.C.Q. (Proc. no. 2010/50445-0) and T.M.S. We thank Dr. Esteban Gonzalez (FuturaGene Corporation) for providing the Eucalyptus A. psidii populations and other facilities as well as Dr. Janice Uchida, Dr. Guus Bakkeren and UNESP/FCAV for providing DNA and urediniospores from different species of Puccinia or Austropuccinia for the specificity assays. We also thank Sarina Tsui and Thiago Gonçalves de Oliveira for helping in field sampling and Dr. Tsai Siu Mui and Dr. Danielle Gregorio Gomes Caldas for providing the structure for the reproducibility tests.

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Correspondence to Carlos Alberto Labate.

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Bini, A.P., Quecine, M.C., da Silva, T.M. et al. Development of a quantitative real-time PCR assay using SYBR Green for early detection and quantification of Austropuccinia psidii in Eucalyptus grandis . Eur J Plant Pathol 150, 735–746 (2018).

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