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

Author(s)

Bini, Andressa Peres ^[1] ; Quecine, Maria Carolina ^[1] ; da Silva, Thalita Moraes ^[1] ; Silva, Luciana Duque ^[2] ; Labate, Carlos Alberto ^[1]

Affiliation

^[1] Univ Sao Paulo, Escola Super Agr Luiz de Queiroz, Dept Genet, Ave Padua Dias 11, BR-13418900 Piracicaba, SP - Brazil

^[2] Univ Sao Paulo, Escola Super Agr Luiz de Queiroz, Dept Ciencias Florestais, Ave Padua Dias 11, BR-13418900 Piracicaba, SP - Brazil

• Document type: Journal article
• Source: European Journal of Plant Pathology; v. 150, n. 3, p. 735-746, MAR 2018.
• Web of Science Citations: 4

Abstract

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. (AU)

• FAPESP's process: 08/50361-1 - Functional genomics applied to the discovery of eucalyptus rust resistance genes
• Grantee: Carlos Alberto Labate
• Support Opportunities: Research Grants - Research Partnership for Technological Innovation - PITE
• FAPESP's process: 13/07596-6 - Molecular study of the pathogenic fungus Puccinia psidii Winter, the causal agent of rust, in vitro and during their interaction with Eucalyptus spp
• Grantee: Andressa Peres Bini
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 14/16804-4 - Deciphering the molecular interaction between Puccinia psidii x Eucalyptus grandis through "omics" approaches
• Grantee: Maria Carolina Quecine Verdi
• Support Opportunities: Research Grants - Young Investigators Grants