Development of a Nested Quantitative Real-Time PCR for DetectingPhytophthora cinnamomiinPersea americanaRootstocks

Abstract

Phytophthora cinnamomi causes Phytophthora root rot (PRR) in avocado (Persea americana), an important disease that causes severe economic losses to the avocado industry globally. To date, no PRR-resistant avocado rootstock variety has been discovered, although certain rootstock varieties have been shown to be more tolerant than others. In this study we developed an accurate, low cost assay for in planta quantification of P. cinnamomi to evaluate disease tolerance. A nested real time PCR assay was developed to sensitively detect pathogen DNA in plant tissues. Root samples from a highly tolerant (Dusa ® ) and less tolerant (R0.12) rootstock were collected at 0, 3, 7, 14 and 21 days after inoculation with P. cinnamomi and used for pathogen quantification. Nested primers developed in this study were specific and sensitive and could detect P. cinnamomi in root tissues. The amount of P. cinnamomi quantified in roots was significantly higher in the less tolerant R0.12 plants when compared to the highly tolerant Dusa ® plants at all time points. This study has confirmed the known status of disease tolerance of Dusa ® and R0.12 avocado rootstocks in a quantitative manner and provides a reliable molecular tool to J. Engelbrecht, 2, Plant Disease assist with industry breeding programs for the selection of PRR-resistant avocado rootstock varieties.

Phytophthora cinnamomi

“…However, the fact that the targeted antigen may not be present in all viable samples can lead to false negative results. DNA, by contrast, is present throughout an organism's lifecycle, and DNA-based assays utilizing qPCR to amplify DNA have a number of advantages over immunodiagnostic techniques (Elliot et al, 2015;Engelbrecht et al, 2013;Eshraghi et al, 2011b;Vincelli and Tisserat, 2008). It should, however, be remembered that assays targeting DNA do not discriminate between viable and non-viable material .…”

Pathways to false positive diagnoses using molecular genetic detection methods; Phytophthora cinnamomi a case study

“…In these assays, the following primers were used: Lpv (N1) (Engelbrecht et al 2013), ITS (N2) (Langrell et al 2011), and ITS (N3) ) ( Table 1). The PCR conditions were as described above and in the original references (Table 1).…”

“…Some primers have been designed for a very specific geographic location and only tested against species thought to occur within that environmental system. For example, Engelbrecht et al (2013) designed primers for use in detecting P. cinnamomi in the avocado industry in South Africa. However, they did not test the primers against P. parvispora, the species most closely related to P. cinnamomi, or P. niederhauseri, both of which are known to occur in South Africa (Oh et al 2013).…”

Diagnóstico molecular de Phytophthora cinnamomi asociado a la pudrición radicular en zonas productoras de aguacate en Ecuador

“…La pudrición radicular causada por Phytophthora cinnamomi Rands es uno de los principales factores que limita la producción del cultivo de aguacate a nivel mundial (Engelbrecht et al 2013;Ochoa et al 2015). Fue identificada por primera vez en Burma (Sumatra) por Rands, en 1922 (Erwin y Ribeiro 1996).…”

“…La tradicional descripción taxonómica de las especies del género Phytophthora se ha basado predominantemente en su morfología, características estructurales del esporangio, anteridio, oogonio o micelio (Hardham 2005;Drenth et al 2006;Engelbrecht et al 2013). En el diagnóstico convencional, se aísla Phytophthora spp., a partir de tejidos enfermos, usando medios de cultivo que contienen un coctel de antibióticos.…”