Development and evaluation of a recombinase polymerase amplification assay for rapid detection of strawberry red stele p
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(2020) 2:26
Phytopathology Research
RESEARCH
Open Access
Development and evaluation of a recombinase polymerase amplification assay for rapid detection of strawberry red stele pathogen Mustafa Ahmad Munawar1* , Anna Toljamo1, Frank Martin2, Elina Oksanen1 and Harri Kokko1
Abstract Phytophthora fragariae causes drastic damage in strawberry crops. P. fragariae infects strawberry roots and causes red stele root rot. Although P. fragariae is a quarantine organism, its spread in Finland continues as more and more fields contract the disease. The spread can be halted through developing rapid and reliable detection assays. We have developed a rapid recombinase polymerase amplification (RPA) assay for P. fragariae targeting the Phytophthora mitochondrial DNA intergenic atp9-nad9 marker. The assay is DNA-extraction free and capable of detecting as low as 10 fg of P. fragariae genomic DNA. We found the assay reliable for diagnosing field plants when samples are adequately collected. We also applied the RPA assay to the detection of the pathogen in the soil through coupling the assay with baiting with the host plant. The results suggest that if only a small number of samples are analysed, the baiting results will not be reliable. Keywords: Phytophthora fragariae, Red stele, Recombinase polymerase amplification, Crude maceration, Baiting, Indicator plants
Background Phytophthora fragariae is a quarantine organism (A2 list of EPPO) and causes red stele root rot of strawberry. This disease is common in regions with cool and moist climates, with damage more extensive in heavy saturated clay soils and during early or late summer. The pathogen first destroys fine roots and later progresses upwards in the stele of larger roots. Plants wilting usually first appears in low or poorly drained areas of fields and the affected area progressively widens over time. When plants with early wilting symptoms are dug out, their major roots have less lateral roots and present a ‘rat-tail appearance’. Dissecting major roots upwards displays abnormal reddish colour in internal cores known as ‘red * Correspondence: [email protected] 1 Department of Environmental and Biological Sciences, University of Eastern Finland, 70211 Kuopio, Finland Full list of author information is available at the end of the article
stele’ and considered as a typical diagnostic feature of the disease. The disease has drastic nature and causes wilting, stunted growth, less or no strawberries and few stolons. Moreover, the pathogen can persist at least ten years in infested soils through its survival form of oospores (Maas, 1998; Ellis, 2008; Newton et al. 2010). The disease has badly damaged strawberry crops in North America, Switzerland, Germany, France and Sweden (Maas 1998). P. fragariae was not present in Finland according to the EPPO/ CABI, 1997. The absence of the pathogen was also confirmed by the countrywide survey for red-stele disease conducted on strawberry roots collected in 1995 (Pohto 1999). However, in 2012 EVIRA (Finnish Food Safety Authority) sur
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