Real-Time PCR Assays for the Detection of Puccinia psidii

Abstract

Puccinia psidii (Myrtle rust) is an emerging pathogen that has a wide host range in the Myrtaceae family; it continues to show an increase in geographic range and is considered to be a significant threat to Myrtaceae plants worldwide. In this study, we describe the development and validation of three novel real-time polymerase reaction (qPCR) assays using ribosomal DNA and β-tubulin gene sequences to detect P. psidii. All qPCR assays were able to detect P. psidii DNA extracted from urediniospores and from infected plants, including asymptomatic leaf tissues. Depending on the gene target, qPCR was able to detect down to 0.011 pg of P. psidii DNA. The most optimum qPCR assay was shown to be highly specific, repeatable, and reproducible following testing using different qPCR reagents and real-time PCR platforms in different laboratories. In addition, a duplex qPCR assay was developed to allow coamplification of the cytochrome oxidase gene from host plants for use as an internal PCR control. The most optimum qPCR assay proved to be faster and more sensitive than the previously published nested PCR assay and will be particularly useful for high-throughput testing and to detect P. psidii at the early stages of infection, before the development of sporulating rust pustules.

Novel PCR Assays for the Detection of Biological Agents Responsible for Wheat Rust Diseases:<b><i> Puccinia triticina</i></b> and <b><i>Puccinia striiformis</i></b> f. sp. <b><i>tritici</i></b>

“…Although the databases for these genes are not abundant, they are useful, especially when variations in the ITS region are insufficient [Gong et al, 2015;Tunarsih et al, 2015]. In the available literature on the detection of Puccinia spp., PCR assays are primarily performed to target tub1 [Baskarathevan et al, 2016;Fraaije et al, 2001;Pan et al, 2010]. In our experiment, the in silico analysis revealed that rpb2 and tub1 have the most unique sites for the detection of Puccinia spp .…”

Chasing myrtle rust in New Zealand: host range and distribution over the first year after invasion

“…Infection with A. psidii was confirmed using a TaqMan real-time PCR assay developed by Baskarathevan et al (2016). The qPCR assay was carried out in a final volume of 20 μl: 10 μl of 2× ToughMix (Quanta Biosciences), a final concentration of 300 nM each PpsiITS1F and PpsiITS1R primer, 120 nM FAM-labelled probe PpsiITS1P, MgCl 2 (4.2 mM final), and 2 μl of DNA template.…”

Estudo molecular do desenvolvimento de <i>Puccinia psidii</i> Winter <i>in vitro</i> e no processo de infecção em<i> Eucalyptus grandis</i>

“…Inicialmente os primers universais CNL12/CNS1, Bt2a/Bt2b e 3F/7R (Tabela (BASKARATHEVAN et al, 2016). No presente trabalho foi possível detectar de 0,5 pg a 5 ng de DNA de P. psidii em E. grandis, confirmando a sensibilidade do conjunto de primers IGS7/IGS9 desenvolvidos.…”

“…Com base nos avanços da biologia molecular, a detecção de P. psidii começou a ser explorada por ensaios de PCR (LANGRELL; GLEN; ALFENAS, 2008;BASKARATHEVAN et al, 2016). Em especial pela técnica de PCR em tempo realqPCR (BASKARATHEVAN et al, 2016) que oferece oportunidade para a rápida detecção e quantificação de agentes patogênicos no interior de seus hospedeiros, auxiliando no diagnóstico de doenças em plantas (YAN et al, 2008;SANZANI et al, 2014;ARIF et al, 2014).…”

“…Em especial pela técnica de PCR em tempo realqPCR (BASKARATHEVAN et al, 2016) que oferece oportunidade para a rápida detecção e quantificação de agentes patogênicos no interior de seus hospedeiros, auxiliando no diagnóstico de doenças em plantas (YAN et al, 2008;SANZANI et al, 2014;ARIF et al, 2014). Devido a utilização de fluorescência, a técnica de qPCR é até 100 vezes mais sensível que o PCR convencional e tem resolução suficiente para detectar um único esporo fúngico (ARIF et al, 2014;PEDLEY, 2009), como já relatado em Glomus intraradices (ALKAN et al, 2004).…”