Quantification of downy mildew (Peronospora sparsa) in Rubus species using real-time PCR

Abstract

Downy mildew disease caused by Peronospora sparsa, also known as ÔdryberryÕ disease, is a serious threat to the cultivation of arctic bramble (Rubus arcticus) and boysenberry (Rubus spp. hybrid). A quantitative and sensitive screening method is necessary for the breeding of downy mildew resistant cultivars and for determining efficient disease control methods. A quantitative real-time PCR method using SYBR Ò Green I fluorescent dye was developed for the analysis of P. sparsa in arctic bramble, other Rubus species and roses. Primers were designed to amplify a P. sparsa specific 94-bp fragment from the internal transcribed spacer region (ITS1) and a 140-bp fragment from a conserved region of plant 5.8S ribosomal DNA, which served as an internal control in the samples. Linear amplification from genomic DNA and control plasmids was achieved with both primers, and even 37 fg of P. sparsa conidial DNA was detected. In the samples collected from the field, quantities as low as 0.2 ppm of P. sparsa DNA in plant DNA were detected, thus enabling the diagnosis of weak and latent infections. Arctic bramble cvs Pima, Mespi and Mesma, all showing distinct foliar symptoms, were tested to assess the relative amount of downy mildew DNA present. The symptoms and the amount of P. sparsa DNA detected correlated only in cv. Pima, indicating that visual inspection of symptoms is not a reliable method for assessing the extent of tissue infection. The number of conidiophores of P. sparsa on in vitro inoculated leaves of two arctic bramble cultivars correlated with the results obtained by real-time PCR.Abbreviations: bp -base pair; Ct -threshold cycle; ITS -internal transcribed spacer; rDNA -ribosomal DNA

Biorational treatments to manage dryberry of blackberry caused by Peronospora sparsa

“…According to Rebollar-Alviter et al (2009), foliar symptoms are not always present, but the absence of these symptoms does not necessarily indicate the absence of disease in the fruit. Similar reports have been published in other growing areas of the world, such as New Zealand (Walter et al, 2014;personal communication); for this reason, diagnosis based on the absence of foliar symptoms is unreliable (Nordskog et al, 2003;Hukkanen et al, 2006Hukkanen et al, , 2008 and should not be the basis for disease management.…”

“…However, the lack of symptoms in the leaves does not indicate an absence of infection, as producers have had significant losses in asymptomatic plantings. Therefore, disease diagnosis based on the presence or absence of foliar symptoms is not a reliable technique for managing this disease (Hukkanen et al, 2006;Rebollar-Alviter et al, 2012). In this regard, different studies have shown that detection based on the Polymerase Chain Reaction (PCR), Real-time PCR and nested PCR using specific primers are more reliable techniques for the rapid and timely detection of the causal agent (Koponen et al, 2000;Hukkanen et al, 2008;RebollarAlviter et al, 2012).…”

Identificación de Peronospora sparsa y evaluación del contenido de fenoles en frutos de mora de castilla afectados por este microorganismo

“…El impacto es considerable, ya que genera pérdidas de producción entre el 50% y 70% (Rodríguez Díaz et al, 2017). Adicionalmente, el uso regular de fungicidas contribuye al desarrollo de cepas resistentes y de difícil control (Hukkanen et al, 2006).…”

PCR-based detection of sunflower white blister rust (Pustula helianthicola C. Rost & Thines) in soil samples and asymptomatic host tissue

“…Wang et al 2006;Pavon et al 2008) or in asymptomatic host tissue (e.g. Aegerter et al 2002;Hukkanan et al 2006). A particular field of interest is the identification of seed contamination with pathogenic oomycetes for which successful approaches have been published for the downy mildew pathogens of basil (Belbahri et al 2005) and sunflower (Thines et al 2004;Ioos et al 2007).…”