Comparison of Five Detection and Quantification Methods for Phytophthora ramorum in Stream and Irrigation Water

Abstract

Propagules of Phytophthora ramorum, the causal agent of sudden oak death (SOD) and ramorum blight, can be recovered from infested stream and nursery irrigation runoff using baiting and filtration methods. Five detection methods, including pear and rhododendron leaf baits, Bottle O’ Bait, filtration, and quantitative polymerase chain reaction (qPCR) performed on zoospores trapped on a filter were compared simultaneously in laboratory assays using lab or creek water spiked with known quantities of P. ramorum zoospores. The detection threshold for each method was determined and methods that could be used to quantify zoospore inoculum were identified. Filtration and qPCR were the most sensitive at detecting low levels of zoospores, followed by wounded rhododendron leaves, rhododendron leaf disks, and pear baits. Filtration, qPCR, and leaf disks were able to quantify P. ramorum zoospores ranging from 2 to 451 direct-plate CFU/liter while wounded leaves and pear baits appeared to be better at detection rather than quantification. The ability to detect and quantify P. ramorum inoculum in water will assist scientists, regulatory agencies, and nursery personnel in assessing the risk of spreading P. ramorum in nurseries and landscape sites where untreated infested water is used for irrigation.

Discriminant haplotypes of avirulence genes of Phytophthora sojae lead to a molecular assay to predict phenotypes

“…Among other applications, they have allowed the precise identification of the causal agents of diseases caused by a complex of fungi (Conti et al ., ), the fulfillment of Koch's postulates with closely related species such as Fusarium spp. (Moine et al ., ), and the diagnosis of the presence and identity of a pathogen from levels that were hitherto undetectable (Rollins et al ., ; Haudenshield et al ., ). In most cases, those PCR‐based assays have exploited conserved regions such as the internal transcribed spacers (ITS) (Bruns and Shefferson, ) or the translation elongation factor 1α (TEF1‐α) to develop diagnostic tools able to identify a given pathogen (Raja et al ., ; Marin‐Felix et al ., ).…”

“…( Moine et al, 2014), and the diagnosis of the presence and identity of a pathogen from levels that were hitherto undetectable (Rollins et al, 2016;Haudenshield et al, 2017). In most cases, those PCR-based assays have exploited conserved regions such as the internal transcribed spacers (ITS) (Bruns and Shefferson, 2004) or the translation elongation factor 1α (TEF1-α) to develop diagnostic tools able to identify a given pathogen (Raja et al, 2017;Marin-Felix et al, 2019).…”

Real-time loop-mediated isothermal amplification: an early-warning tool for quarantine plant pathogen detection

“…For these reasons, sensitive and specific tools for effective phytosanitary inspection and interception are required to prevent new pathogen introductions. Nowadays, the high specificity and sensitivity of molecular DNA-based technologies allows detection of pathogens in the early stages of infection, when they are present at low DNA concentrations (Bilodeau et al 2007; Chandelier et al 2006; Harper et al 2010; Luchi et al 2013; Rollins et al 2016). Although many of these methods have been used routinely in the laboratory, most of them are not transferable for field inspection, seriously limiting their adequacy for point-of-care application (Lau and Botella 2017).…”

Transport Mechanisms of Motile and Nonmotile Phytophthora cactorum Zoospores in Unsaturated Porous Media

“…An aliquot of sand (0.5 g) was taken from the column at 0, 3, 6, 9, 12, and 15 cm depth and placed into Petri dishes filled with PARPH‐V8 medium. The PARPH‐V8 medium was prepared following published protocols (Ferguson & Jeffers, 1999; Rollins et al., 2016), and consisted of one‐third V8 agar amended with 10 mg of Delvocid (50% pimaricin), 250 mg of sodium ampicillin, 10 mg of Rifamycin‐SV (sodium salt), 66.7 mg of Terraclor (75% pentachloronitrobenzene), and 50 mg of Hymexazol. Then, 1 mL background solution was added to evenly distribute sand grains on the PARPH‐V8 medium (Wilkinson et al., 1981).…”

“…In the soil columns, the retained zoospores could not be measured with either UV‐Vis spectrophotometry or colony counting because of substantial background noise; thus, we used the leaf baiting method to quantify the amount of retained zoospores (Rollins et al., 2016). Bait leaves were harvested from field‐grown Rhododendron “Nova Zembla” at the WSU Puyallup Research and Extension Center (Puyallup, WA), cleaned with 0.5% hypochlorite solution and deionized water, and cut into 6‐mm diameter round baits using a cork borer (Rollins et al., 2016). Soil was excavated in 1‐cm depth increments, and an aliquot (10 g) of wet soil was placed into a 50 mL centrifuge tube.…”