Temporal patterns of airborne Phytophthora spp. in a woody plant nursery area detected using real-time PCR

An effective framework for early warning and rapid response is a crucial element to prevent or mitigate the impact of biological invasions of plant pathogens, especially at ports of entry. Molecular detection of pathogens by using PCR-based methods usually requires a well-equipped laboratory. Rapid detection tools that can be applied as point-of-care diagnostics are highly desirable, especially to intercept quarantine plant pathogens such as Xylella fastidiosa , Ceratocystis platani and Phytophthora ramorum , three of the most devastating pathogens of trees and ornamental plants in Europe and North America. To this aim, in this study we developed three different loop mediated isothermal amplification (LAMP) assays able to detect each target pathogen both in DNA extracted from axenic culture and in infected plant tissues. By using the portable instrument Genie ® II, the LAMP assay was able to recognize X. fastidiosa , C. platani and P. ramorum DNA within 30 min of isothermal amplification reaction, with high levels of specificity and sensitivity (up to 0.02 pg µL −1 of DNA). These new LAMP-based tools, allowing an on-site rapid detection of pathogens, are especially suited for being used at ports of entry, but they can be also profitably used to monitor and prevent the possible spread of invasive pathogens in natural ecosystems.

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“…Real time PCR protocols for C. platani and P. ramorum were those described in Luchi et al (2013) and Migliorini et al (2018), respectively.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2019

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“…Although spore traps have been largely used in epidemiology, so far, most studies have been performed at local scale (i.e., with spore traps usually at ground level, within or in the proximity of forest stands or crop fields). The few studies using air samplers set up on rooftops or towers for the detection of plant pathogens were generally performed at only a few sites and dates, and none reported the detection of forest pathogens (Brittain et al ., 2013; Banchi et al ., 2018; Migliorini et al ., 2019). Our study takes a step further by clearly positioning this approach in an epidemiological surveillance perspective using an established network of traps, such as those operated for pollen monitoring, and by providing data that demonstrate its feasibility and applicability for forest pathogens.…”

Section: Discussionmentioning

confidence: 99%

“…The first issue was to confirm that forest pathogenic fungi can be detected in aerobiological samples from pollen monitoring networks, despite the location of traps on rooftops in urban environments (providing regional sampling but with probably very low densities of spores). Although a few studies report the detection of some crop pathogens in rooftop traps (Brittain et al, 2013;Banchi et al, 2018;Migliorini et al, 2019), they do not document the presence of forest pathogens. To answer this question, we used two molecular detection methods, a metabarcoding approach and specific real-time PCR.…”

Section: Introductionmentioning

confidence: 99%

Combining permanent aerobiological networks and molecular analyses for large‐scale surveillance of forest fungal pathogens: A proof‐of‐concept

Aguayo¹,

Husson

Chancerel

et al. 2020

Plant Pathology

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Forest disease management relies principally on a preventive approach in which epidemiological surveillance plays a crucial role. However, efficient and cost-effective surveillance methods are not currently available for large spatial scales. Nevertheless, aerobiological networks have been set up for several decades in many countries to monitor pollen dispersal and provide real-time assessments of allergenic risk. Here, we suggest that the same approach could be used for the surveillance of forest pathogens. Using molecular methods, we analysed samples from 12 sites of the French aerobiological network, at different dates. Both metabarcoding by high-throughput sequencing (using two markers and two different bioinformatics approaches) and real-time PCR targeting eight important forest pathogens were conducted. To validate the approach, temporal and spatial trends of spore detection were compared with field disease data. The metabarcoding approach demonstrated that many fungal plant pathogens could be found in aerobiological samples. Moreover, five of the eight targeted forest pathogens were detected by real-time PCR, with temporal and spatial trends of spore capture consistent with field data. In particular, Hymenoscyphus fraxineus was detected at high frequency in aerobiological samples in the areas where ash dieback has been present for the longest period of time, and at lower frequency in areas with more recent invasion. Spore detection of seasonal pathogens showed a temporal pattern similar to that of disease reports. Overall, our study provides a proof of concept that permanent aerobiological networks combined with molecular methods may provide a useful tool for large-scale surveillance of forest pathogens.

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“…New opportunities to combine air sampling with qPCR to identify and quantify fungal pathogens have been developed for Caliciopsis pinea, C. platani, F. circinatum, H. fraxineus, and Phytophthora spp. (Botella et al 2019;Chandelier et al 2010Chandelier et al , 2014Luchi et al 2013;Migliorini et al 2019;Schweigkofler et al 2004). The use of these techniques can provide more accurate forecasts of the risk of pathogen spread, and help the management of the disease.…”

Section: Disease Controlmentioning

confidence: 99%

Fast and reliable molecular methods to detect fungal pathogens in woody plants

Luchi

Ioos

Santini

2020

Appl Microbiol Biotechnol

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Plant diseases caused by pathogenic microorganisms represent a serious threat to plant productivity, food security, and natural ecosystems. An effective framework for early warning and rapid response is a crucial element to mitigate or prevent the impacts of biological invasions of plant pathogens. For these reasons, detection tools play an important role in monitoring plant health, surveillance, and quantitative pathogen risk assessment, thus improving best practices to mitigate and prevent microbial threats. The need to reduce the time of diagnosis has prompted plant pathologists to move towards more sensitive and rapid methods such as molecular techniques. Considering prevention to be the best strategy to protect plants from diseases, this review focuses on fast and reliable molecular methods to detect the presence of woody plant pathogens at early stage of disease development before symptoms occur in the host. A harmonized pool of novel technical, methodological, and conceptual solutions is needed to prevent entry and establishment of new diseases in a country and mitigate the impact of both invasive and indigenous organisms to agricultural and forest ecosystem biodiversity and productivity.

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Temporal patterns of airborne Phytophthora spp. in a woody plant nursery area detected using real-time PCR

Cited by 10 publications

References 54 publications

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

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

Combining permanent aerobiological networks and molecular analyses for large‐scale surveillance of forest fungal pathogens: A proof‐of‐concept

Fast and reliable molecular methods to detect fungal pathogens in woody plants

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