A Comparative Analysis of Detection Techniques Used in US Regulatory Programs to Determine Presence of <i>Phytophthora ramorum</i> in <i>Camellia japonica</i> ‘Nucio's Gem’ in an Infested Nursery in Southern California

Bulluck, Russ; Shiel, P. J.; Berger, P. H.; Kaplan, D. T.; Parra, Greg; Li, Wenbin; Lévy, L.; Keller, John D.; Sharma, Nawal; Dennis, Michelle M.; Stack, James P.; Pierzynski, Joy; O'Mara, J. K.; Webb, Craig A.; Finley, Ledare; Lamour, Kurt; McKemy, J. M.; Palm, Mary E.

doi:10.1094/php-2006-1016-01-rs

Cited by 16 publications

(7 citation statements)

References 3 publications

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“…Seedlings were hand-planted; in mulch treatments, seedlings were placed with the crowns above the soil line to encourage root growth within the mulch. However, studies with P. ramorum in leaf tissue showed a false negative rate of 29% for this ELISA test kit and 56% for culturing, as compared with detection using nested PCR (12), indicating that a 91% recovery rate may be taken as acceptable confirmation of Phytophthora infection in the symptomatic plants. Chemical mowing (low rates of glyphosate) was used for weed control, with some addifional mowing and handweeding performed during plot assessments.…”

Section: Methodsmentioning

confidence: 89%

Microbial Profiling of Cultural Systems for Suppression of Phytophthora Root Rot in Fraser Fir

2011

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Richter, B. S., Benson, D. M., and Ivors. K. L. 2011. Microbial profiling of cultural systems for suppression of Phytophthora root rot in Fraser ftr. Plant Dis. 95:537-546.Phytophthora root rot of Fraser ftr. caused by several Phytophthora spp., is a severe problem in Christmas tree production. Since fungicides are not economically viable for disease management in fteld plantings and host resistance is not available, cultural control methods were investigated. Mulches, dairy compost, and soil pH adjustment were tested at ftve fteld sites in North Carolina. Treatments included wood chips, wood chips plus compost, or pine bark as raised beds, and cotnpost or sulfur tilled into soil. Soil and mulch microbial populations were characterized by dilution plating and calculation of a log series diversity index, and by enzyme analyses at 5, 12. 17. and 24 months after planting. Bacterial and fungal counts, microbial activity, and cellulase activity were higher in mulch than in soil at all sites and times {P < 0.01). and generally did not differ among mulch types or atnong soils. Treatments significantly affected disease ratings and tree survival at three of ftve sites, with one or more mulch treatments yielding lower disease ratings and greater survival than controls. Tree mortality at each time point varied significantly with cellula.se activity in the upper root zone {P = 0.005). Other biological variables did not show significant relationships with disease ratings or mortality.

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Section: Methodsmentioning

confidence: 89%

Microbial Profiling of Cultural Systems for Suppression of Phytophthora Root Rot in Fraser Fir

2011

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“…This result may be due to coalescing foci, as suggested by a comparative analysis of different detection techniques (above-and below-ground ELISA, real-time-PCR, nested PCR and culture) in a P. ramorum-infested nursery growing Camellia japonica in California (Bulluck et al, 2006).…”

Section: Prevention Of Infestation Of the Commoditymentioning

confidence: 99%

“…Although a preliminary study from California suggests a spread in nurseries that is within the range of what is imposed by regulations (Tjosvold, 2010), Heungens et al (2010) reported no clear pattern of focal spread, as the same genotype was detected at more than 10 m distances in a genetic study over 2 growing seasons at a large commercial nursery in Belgium with multiple infestations of P. ramorum. This result may be due to coalescing foci, as suggested by a comparative analysis of different detection techniques (above-and below-ground ELISA, real-time-PCR, nested PCR and culture) in a P. ramorum-infested nursery growing Camellia japonica in California (Bulluck et al, 2006).…”

Section: • Prevention Of Infestation Of the Commoditymentioning

confidence: 99%

Scientific Opinion on the Pest Risk Analysis onPhytophthora ramorumprepared by the FP6 project RAPRA

Rossi¹

2011

EFSA Journal

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The Panel on Plant Health was asked to deliver a scientific opinion on the Pest Risk Analysis on Phytophthora ramorum prepared by the FP6 project RAPRA, taking into account comments by Member States and additional information since RAPRA. P. ramorum is the oomycete causing sudden oak death in the USA and leaf and twig blight/dieback on a range of ornamental species in North America and Europe. Currently P. ramorum is not listed as a harmful organism in Council Directive 2000/29/EC, but the Commission adopted in 2002 provisional emergency measures to prevent introduction into and spread within the EU. Recent large-scale outbreaks in Japanese larch (Larix kaempferi) plantations in the UK and Ireland have worsened the potential consequences in the risk assessment area. However, the Panel concludes that the broad narrative in the RAPRA report stands and supports its conclusion that "There is a risk of further entry (of known or new lineages and/or mating types), establishment and […] impact". It is advisable to avoid introductions of different lineages because of inherent phenotypic differences and the potential for sexual recombination. The Panel supports the management options proposed in the RAPRA report and adds further measures for consideration. Uncertainty remains over the extent to which the association between control measures and gradual reduction in the number of cases in nurseries is causal. The emergency measures have not prevented outbreaks occurring in the natural environment. The many other remaining uncertainties (fitness of progeny, hybridisation with other Phytophthora species, host range and epidemiological role of new hosts, early detection of new outbreaks, understanding of long-range dispersal, structure of plant trade networks, origin of the pathogen) call for further research on P. ramorum across Europe. 4 Editorial changes have been made on pages 51 (acronym PRA removed as it was not in line with the Panel's dictionary as discussed in Appendix 2 of the Guidance on a harmonised framework for pest risk assessment and the identification and evaluation of pest risk management options by EFSA) and 89 (statement which referred to the Panel agreeing with the RAPRA conclusion that "P. ramorum fulfils the criteria of a quarantine pest" was removed as requested by the Panel during adoption of the draft opinion). The changes do not affect the overall conclusions of the opinion. To avoid confusion the original version has been removed from the website. The scope of this Scientific Opinion is the evaluation of the RAPRA report, taking into consideration the comments of Member States as well as additional information published after finalisation of the RAPRA report, or not cited in the RAPRA report. A systematic literature search until March 2011 was carried out. It should be noted that the scientific community is concerned by the shift of P. ramorum to new hosts (with a special focus on the Japanese larch) and its further spread, and therefore new results of ongoing research are constantly be...

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“…These diseases can be difficult or impossible to distinguish by symptoms alone and in-lab diagnostic testing is required for accurate pathogen identification. Identification of ambiguous Phytophthora species has traditionally relied on techniques such as baiting, isolation onto a Phytophthora semi-selective medium, DNA extraction and polymerase chain reaction (PCR) or antibodies (i.e., immunostrips) to identify the genus or species present [5][6][7][8]. However, this is time consuming and some species, such as the causal agent of sudden oak death, Phytophthora ramorum [9], are of regulatory importance, requiring a rapid and accurate identification.…”

Section: Introductionmentioning

confidence: 99%

Validation of a Preformulated, Field Deployable, Recombinase Polymerase Amplification Assay for Phytophthora Species

McCoy

Miles

Bilodeau

et al. 2020

Plants

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Recombinase polymerase amplification (RPA) assays are valuable molecular diagnostic tools that can detect and identify plant pathogens in the field without time-consuming DNA extractions. Historically, RPA assay reagents were commercially available as a lyophilized pellet in microfuge strip tubes, but have become available in liquid form more recently—both require the addition of primers and probes prior to use, which can be challenging to handle in a field setting. Lyophilization of primers and probes, along with RPA reagents, contained within a single tube limits the risk of contamination, eliminates the need for refrigeration, as the lyophilized reagents are stable at ambient temperatures, and simplifies field use of the assays. This study investigates the potential effect of preformulation on assay performance using a previously validated Phytophthora genus-specific RPA assay, lyophilized with primers and probes included with the RPA reagents. The preformulated lyophilized Phytophthora RPA assay was compared with a quantitative polymerase chain reaction (qPCR) assay and commercially available RPA kits using three qPCR platforms (BioRad CFX96, QuantStudio 6 and Applied Biosystems ViiA7) and one isothermal platform (Axxin T16-ISO RPA), with experiments run in four separate labs. The assay was tested for sensitivity (ranging from 500 to 0.33 pg of DNA) and specificity using purified oomycete DNA, as well as crude extracts of Phytophthora-infected and non-infected plants. The limit of detection (LOD) using purified DNA was 33 pg in the CFX96 and ViiA7 qPCR platforms using the preformulated kits, while the Axxin T16-ISO RPA chamber and the QuantStudio 6 platform could detect down to 3.3 pg with or without added plant extract. The LOD using a crude plant extract for the BioRad CFX96 was 330 pg, whereas the LOD for the ViiA7 system was 33 pg. These trials demonstrate the consistency and uniformity of pathogen detection with preformulated RPA kits for Phytophthora detection when conducted by different labs using different instruments for measuring results.

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A Comparative Analysis of Detection Techniques Used in US Regulatory Programs to Determine Presence of Phytophthora ramorum in Camellia japonica ‘Nucio's Gem’ in an Infested Nursery in Southern California

Cited by 16 publications

References 3 publications

Microbial Profiling of Cultural Systems for Suppression of Phytophthora Root Rot in Fraser Fir

Microbial Profiling of Cultural Systems for Suppression of Phytophthora Root Rot in Fraser Fir

Scientific Opinion on the Pest Risk Analysis onPhytophthora ramorumprepared by the FP6 project RAPRA

Validation of a Preformulated, Field Deployable, Recombinase Polymerase Amplification Assay for Phytophthora Species

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