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Pyricularia oryzae is a fungal plant pathogen causing blast disease in several species of the Poaceae family. It encompasses several genetic lineages, including one that is pathogenic on wheat and belongs to the Triticum lineage of P. oryzae. The fungus spreads at short distances by its airborne and rain-splash dispersed spores, and at longer distances via cryptically infected wheat seeds, through trade. Here, we describe a practical method to detect P. oryzae Triticum lineage in wheat seeds, after a biological enrichment step, with various options for molecular testing involving several DNA-based technologies: polymerase chain reaction (PCR), real-time PCR, and loop-mediated isothermal amplification (LAMP). The array of available molecular assays is presented in this protocol, each of them targeting specific regions of the P. oryzae Triticum lineage and offering different levels in terms of sensitivity and specificity.

Phyllosticta citricarpa, Elsinoe fawcettii, Elsinoe australis, and Pseudocercospora angolensis are major pathogens of citrus crops worldwide and can cause non-characteristic symptoms that may lead to confusion regarding the causative agent. These fungi are subject to international phytosanitary regulations, and testing on fruits or leaves requires accurate and easy-to-use tools. New multiplex conventional PCR and real-time PCR assays were developed here to achieve highly accurate simultaneous detection of all four fungal pathogens in fruit tissues. We designed new oligonucleotide combinations for P. citricarpa, E. fawcettii, and E. australis and combined them with already available primers and hydrolysis probes to be used in either PCR assay. The limit of detection for multiplex conventional PCR was as low as 100 pg μL−1 for P. citricarpa, E. fawcettii, and E. australis and 10 pg μL−1 of target DNA per reaction tube for P. angolensis. The quadruplex real-time PCR assay successfully yielded repeatable positive results with as low as 242, 243, 241, and 242 plasmidic copies of target DNA of P. citricarpa, E. fawcettii, E. australis, and P. angolensis, respectively. Moreover, analysis of 60 naturally infected citrus samples yielded 100% concordant results by both assays. Our validation experiment revealed that the multiplex real-time PCR assay showed high specificity except a cross-reaction with P. paracitricarpa DNA. Sensitivity, repeatability, reproducibility, and robustness were verified, and the assay could be used following different DNA extraction procedures, supporting fitness for routine analysis. These new multiplex tools should be of great interest as cost-effective solutions for regulatory authorities and diagnostic laboratories, enabling testing for four important pathogens in single-tube reactions. • Development of new conventional PCR and qPCR assays for four citrus pathogens. • Very low limits of detection were found for multiplex conventional PCR. • qPCR had high specificity, sensitivity, repeatability, reproducibility, and robustness.

Many species of fungal plant pathogens coexist as multiple lineages on the same host, but the factors underlying the origin and maintenance of population structure remain largely unknown. The rice blast fungus Pyricularia oryzae is a widespread model plant pathogen displaying population subdivision. However, most studies of natural variation in P. oryzae have been limited in genomic or geographic resolution, and host adaptation is the only factor that has been investigated extensively as a contributor to population subdivision. In an effort to complement previous studies, we analyzed genetic and phenotypic diversity in isolates of the rice blast fungus covering a broad geographical range. Using single-nucleotide polymorphism genotyping data for 886 isolates sampled from 152 sites in 51 countries, we showed that population subdivision of P. oryzae in one recombining and three clonal lineages with broad distributions persisted with deeper sampling. We also extended previous findings by showing further population subdivision of the recombining lineage into one international and three Asian clusters, and by providing evidence that the three clonal lineages of P. oryzae were found in areas with different prevailing environmental conditions, indicating niche separation. Pathogenicity tests and bioinformatic analyses using an extended set of isolates and rice varieties indicated that partial specialization to rice subgroups contributed to niche separation between lineages, and differences in repertoires of putative virulence effectors were consistent with differences in host range. Experimental crosses revealed that female sterility and early post-mating genetic incompatibilities acted as strong additional barriers to gene flow between clonal lineages. Our results demonstrate that the spread of a fungal pathogen across heterogeneous habitats and divergent populations of a crop species can lead to niche separation and reproductive isolation between distinct, widely distributed, lineages.

Producción Científica, Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii, causing cankers in trees of all ages, damping-of in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F. circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F. circinatum and related Fusarium species. Diagnostic sensitivity, specifcity and accuracy of the nine protocols all reached values >80%, and the diagnostic specifcity was the only parameter difering signifcantly between protocols.The rates of false positives and of false negatives were computed and only the false positive rates difered signifcantly, ranging from 3.0% to 17.3%.The diference between protocols for some ofthe performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F. circinatum were not easily transferable to endusers. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data priorto use under modifed conditions (i.e. reagents and equipment). Suggestions to improve the transfer are proposed., European Cooperation in Science and Technology (COST Action FP1406 PINESTRENGTH), Estonian Science Foundation (grants PSG136 and IUT21-04), Centro de Estudios do Ambiente e do Mar (CESAM) (projects UID/AMB/50017/2013 and POCI-01- 0145- FEDER-007638), Slovenian Research Agency (project P4-0107)

International audience; 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.

Producción Científica, Fusarium circinatum, the causal agent of pine pitch canker (PPC), is currently one of the most important threats of Pinus spp. globally. This pathogen is known in many pine-growing regions, including natural and planted forests, and can affect all life stages of trees, from emerging seedlings to mature trees. Despite the importance of PPC, the global distribution of F. circinatum is poorly documented, and this problem is also true of the hosts within countries that are affected. The aim of this study was to review the global distribution of F. circinatum, with a particular focus on Europe. We considered (1) the current and historical pathogen records, both positive and negative, based on confirmed reports from Europe and globally; (2) the genetic diversity and population structure of the pathogen; (3) the current distribution of PPC in Europe, comparing published models of predicted disease distribution; and (4) host susceptibility by reviewing literature and generating a comprehensive list of known hosts for the fungus. These data were collated from 41 countries and used to compile a specially constructed geo-database. A review of 6297 observation records showed that F. circinatum and the symptoms it causes on conifers occurred in 14 countries, including four in Europe, and is absent in 28 countries. Field observations and experimental data from 138 host species revealed 106 susceptible host species including 85 Pinus species, 6 non-pine tree species and 15 grass and herb species. Our data confirm that susceptibility to F. circinatum varies between different host species, tree ages and environmental characteristics. Knowledge on the geographic distribution, host range and the relative susceptibility of different hosts is essential for disease management, mitigation and containment strategies. The findings reported in this review will support countries that are currently free of F. circinatum in implementing effective procedures and restrictions and prevent further spread of the pathogen., COST Action FP1406 (PINESTRENGTH), the Estonian Science Foundation grant PSG136, the Forestry Commission, United Kingdom, the Phytophthora Research Centre Reg. No. CZ.02.1.01/0.0/0.0/15_003/0000453, a project co-financed by the European Regional Development Fund., French National Research Agency (ANR) (ANR-11-LABX-0002-01), BBSRC, Defra, ESRC, the Forestry Commission, NERC and the Scottish Government (BB/L012251/1)

Many invasive fungal species coexist as multiple lineages on the same host, but the factors underlying the origin and maintenance of population structure remain largely unknown. Here, we analyzed genetic and phenotypic diversity in isolates of the rice blast fungus (Pyricularia oryzae) covering a broad geographical range. We showed that the four lineages of P. oryzae were found in areas with different prevailing environmental conditions and types of rice grown, indicating niche separation. Pathogenicity tests revealed that specialization to rice subspecies contributed to niche separation between lineages, and differences in repertoires of putative virulence effectors were consistent with differences in host range. Experimental crosses revealed that female sterility and early post-mating genetic incompatibilities acted as strong barriers to gene flow between these lineages. Our results demonstrate that the spread of a pathogen across heterogeneous habitats and divergent populations of a crop species can lead to niche separation and reproductive isolation between distinct invasive lineages.

Wheat blast is a devastating disease caused by the pathogenic fungus Pyricularia oryzae. Wheat blast first emerged in South America before more recently reaching Bangladesh. Even though the pathogen can spread locally by air-dispersed spores, long-distance spread is likely to occur via infected wheat seed or grain. Wheat blast epidemics are caused by a genetic lineage of the fungus, called the Triticum lineage, only differing from the other P. oryzae lineages by less than 1% genetic divergence. In order to prevent further spread of this pathogen to other wheat-growing areas in the world, sensitive and specific detection tools are needed to test for contamination of traded seed lots by the P. oryzae Triticum lineage. In this study, we adopted a comparative genomics approach to identify new loci specific to the P. oryzae Triticum lineage and used them to design a set of new markers that can be used in conventional polymerase chain reaction (PCR), real-time PCR, or loop-mediated isothermal amplification (LAMP) for the detection of the pathogen, with improved inclusivity and specificity compared to currently available tests. A preliminary biological enrichment step of the seeds was shown to improve the sensitivity of the tests, which enabled the detection of the target at an infection rate as low as 0.25%. Combined with others, this new toolkit may be particularly beneficial in preventing the trade of contaminated seeds and in limiting the spread of the disease.

Black spot symptoms were reported on vanilla plants in Reunion Island in 2011. They have repeatedly reduced annual pod production by 10% to 30%. The disease is characterized by dark spots that appear in slight depressions on flowers, pods, leaves and stems. The spots then develop into broad clearly depressed necrotic plaques. Morphological and molecular analyses, as well as pathogenicity tests, identified the fungus Colletotrichum orchidophilum (Ascomycota) as the causal agent of the disease. Inoculation tests in controlled conditions confirmed that the two C. orchidophilum strains isolated from fruit lesions are pathogenic on the leaves and fruits of Vanilla planifolia (accessions CR0001 and CR0020). However, these strains induced symptoms only when the epidermis of leaves and fruits had been punctured by a needle. In the absence of injury, no symptom appeared. Colletotrichum arxii and Fusarium proliferatum (Ascomycota) are fungal species that are also frequently isolated from black spot lesions. However, they are not pathogenic to vanilla. This is the first report of C. orchidophilum in Reunion Island. It is also the first demonstration of C. orchidophilum's pathogenicity to an orchid. Simple preventive control measures were proposed to reduce the incidence of black spot disease in vanilla plots.

International audience; Fusarium head blight (FHB) is a cereal disease caused by a complex of plant pathogenic fungal species of the genera Fusarium andMicrodochium, with different epidemiological characteristics and producing or not different toxins. The FHB causes significant yieldlosses, health issues and technological quality concerns in all temperate areas worldwide. The composition of the species complexis different according to the cereal, depending on the climate of the year and the agronomic practices. These species may havedifferent susceptibilities to the fungicides used and different behaviors towards genetic resistance. Thus, investigating thesecharacteristics is required to implement integrated pest management strategies adapted to this disease. For more than 10 years,several research programs have led to significant progress in this area. Tools have been developed to more accurately characterizefungal populations and their interactions. Models have been developed to predict the risk and to adapt control measures. Themolecular mechanisms involved in the interaction between plant genes and the pathogenicity of fungal species are beginning to bedeciphered. Phenotyping methods are being developed to help in the characterization of varietal susceptibility and direct controlmethods using biocontrol products are under investigation. The goal of this paper is to review the main results obtained in thecontext of integrated management of fusarium head blight.; La fusariose de l’épi (FHB) est une maladie des céréales causée par un complexe d’espèces de champignons phytopathogènes desgenres Fusarium et Microdochium, ayant des caractéristiques épidémiologiques différentes et productrices ou non de différentestoxines. Le FHB entraîne d’importantes pertes de rendement et des problèmes de qualité sanitaire et technologique dans toutes lesrégions tempérées du globe. La composition du complexe d’espèces est différente selon les céréales, très dépendante du climat del’année et de l’itinéraire technique. Ces espèces peuvent avoir des sensibilités différentes aux fongicides utilisés et descomportements différents vis à vis des résistances présentes dans les variétés. Ainsi, il est essentiel de connaître ces caractéristiquespour mettre en œuvre les méthodes de lutte intégrée adaptées à cette maladie. Depuis plus de 10 ans, plusieurs programmes derecherche ont permis des avancées significatives sur cette thématique. Des outils ont été développés et ont permis de caractériserplus précisément les populations fongiques selon les céréales et leurs interactions. Des modèles ont été développés pour prédire lerisque et adapter les moyens de lutte. Les mécanismes moléculaires impliqués dans l’interaction entre les gènes de la plante et lapathogénicité des espèces fongiques commencent à être décryptés. Des méthodes de phénotypage se développent pour aider à lacaractérisation de la sensibilité variétale et des méthodes de lutte directe utilisant des produits de biocontrôle sont endéveloppement. L’objectif de cet article est de synthétiser les principaux résultats acquis dans l’objectif d’une protection intégréevis‐à‐vis de la fusariose des épis des céréales.

International audience; Rapid detection is key to managing emerging diseases because it allows their spread around the world to be monitored and limited. The first major wheat blast epidemics were reported in 1985 in the Brazilian state of Paraná. Following this outbreak, the disease quickly spread to neighboring regions and countries and, in 2016, the first report of wheat blast disease outside South America was released. This Asian outbreak was due to the trade of infected South American seed, demonstrating the importance of detection tests in order to avoid importing contaminated biological material into regions free from the pathogen. Genomic analysis has revealed that one particular lineage within the fungal species Pyricularia oryzae is associated with this disease: the Triticum lineage. A comparison of 81 Pyricularia genomes highlighted polymorphisms specific to the Triticum lineage, and this study developed a real-time PCR test targeting one of these polymorphisms. The test's performance was then evaluated in order to measure its analytical specificity, analytical sensitivity, and robustness. The C17 quantitative PCR test detected isolates belonging to the Triticum lineage with high sensitivity, down to 13 plasmid copies or 1 pg of genomic DNA per reaction tube. The blast-based approach developed here to study P. oryzae can be transposed to other emerging diseases. © 2020 The American Phytopathological Society

Rapid detection is key to managing emerging diseases because it allows their spread around the world to be monitored and limited. The first major wheat blast epidemics were reported in 1985 in the Brazilian state of Paraná. Following this outbreak, the disease quickly spread to neighboring regions and countries and, in 2016, the first report of wheat blast disease outside South America was released. This Asian outbreak was due to the trade of infected South American seed, demonstrating the importance of detection tests in order to avoid importing contaminated biological material into regions free from the pathogen. Genomic analysis has revealed that one particular lineage within the fungal species

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.

International audience; 1. Ash dieback is induced by Hymenoscyphus fraxineus, an invasive pathogenic fungus. It is causing severe damage to European ash populations. However, the local environment, such as climate or site conditions, is known to affects ash dieback.2. We studied the landscape epidemiology of the disease on a 22 km² area in northeastern France at two stages of the invasion process using Bayesian spatio-temporal models fitted with integrated nested Laplace approximation (INLA). Several features characterizing disease severity, crown dieback, frequency of collar canker, and density of infected leaf debris in the litter were determined on a regular grid over a 3.5 x 6.5 km area. We first analysed the effect of landscape features on the disease establishment stage in 2012, two years after the first report of the disease in the area, and then on further disease development, in 2016-2018. 3.Landscape features had little impact on the disease at the establishment stage, but strongly determined its further development. Local fragmentation of tree cover was the most important factor, with trees that are isolated or in hedges far less affected than trees in a forest environment. We showed that they were subjected to different microclimates, with higher crown temperatures unfavourable to pathogen development. Low host density strongly reduced disease development. The presence of large ash populations in the vicinity affected local disease severity up to several hundred meters. 4. Synthesis. We showed that the landscape characteristics strongly affect the development and spread of ash dieback. The disease is far less severe in forest conditions when ash density is low or in open canopies such as hedges and isolated trees. Ash trees are often in these types of landscapes, which should strongly limit the overall impact of Ash dieback.

Podosphaera pannosa (Wallr.:Fr.) de Bary (anamorph Oïdium leucoconium Desm.) is described as the most frequent species causing powdery mildew of members of the Rosaceae family, especially on Rosa spp. and Prunus spp. P. pannosa is reported as cosmopolitan, but its occurrence on Prunus cerasus (cherry) is limited to Hungary (3). During spring 2011, typical symptoms of powdery mildew were observed in a Prunus cerasus orchard located in La Roche de Glun (southeastern France). On average, 25% of the shoots per individual tree were affected by this disease. Although several different cultivars were grown in the orchard, cultivar Bigalise alone displayed powdery mildew symptoms. The lower surface of the leaves was covered with superficial, white, dense mycelium, whereas the upper side showed discoloration, necrosing patches, and blisters. Microscopic slides were prepared from fresh material by gently pressing a clear adhesive tape onto the lower surface covered by mycelium, which was further stained with lactic acid/methyl blue. The presence of powdery mildew was confirmed by the observation of typical microscopic features of the anamorphic stage of the fungus (2). Conidiophores were erect. Conidia (oïdia) were hyaline and keg-shaped, and developed basipetally in chains of six to eight conidia. Conidial dimensions were 17 to 29 (23) × 9 to 17 (14) μm. No cleistothecia (teleomorphic state of the fungus) were observed. Species identity was determined by sequencing the ITS region of the rDNA followed by comparison with reference sequences available on GenBank (1). Fungal material was collected from infected leaves by scraping the mycelium with a sterile needle, and was transferred into 2-ml microtubes. Fungal total DNA was then extracted using a commercial plant DNA extraction kit and the ITS region was amplified by PCR using the ITS1-ITS4 primer pair (4). Nucleotide sequence was determined and deposited in GenBank (Accession No. JN654341). Analysis of the sequence by BLAST showed 100% identity with Podosphaera pannosa. To our knowledge, this is the first report of Podosphaera pannosa on Prunus cerasus in France. This species was hitherto scarcely reported on cherry trees, and may deserve more attention in the future. References: (1) M. Gàbor et al. Eur. J. Plant Pathol. 131:135, 2011. (2) G. Grove et al. Page 12 in: Compendium of Stone Fruit Diseases. American Phytopathological Society, St Paul, MN, 1995. (3) L. Vajna et al. New Dis. Rep. 12:15, 2005. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications, 1990.

Cylindrocladium buxicola Henricot causes twig blight on Buxus spp., severe defoliation, and eventually death of plants, especially in young seedlings (1). The disease was first observed in the United Kingdom and New Zealand in the 1990s and recently, the fungus was detected in other European countries (1). In November 2006, box blight symptoms were observed in a Buxus sempervirens nursery located in the South West of France (La Reole). Since then, more diseased samples from other French sites, including forestry areas and ornamental garden nurseries, have been received, indicating that this disease is spreading. Symptomatic twig samples were sent for lab analysis and dark brown spots were observed on the leaves, sometimes coalescing to cover the entire leaf surface, with black streaks on the stems. Fungal fruiting structures were observed directly on the leaf surface and were examined with a stereomicroscope. Microscopic slides were then prepared by gently pressing a clear adhesive tape onto the surface covered by mycelium and spores, which was further stained with lactic acid and methyl blue. Cylindrical, straight, biseptate, hyaline conidia, 53.8 to 75.3 (64.4) × 4.4 to 5.2 (4.6) μm, were observed, sterile hyphae with terminal vesicles ended with a pointed apex, and conidiophores were penicilliate; all of those characters were consistent with C. buxicola (2). To support the diagnosis, fruiting structures were plated on malt agar media supplemented with 100 ppm of chloramphenicol. The pure culture obtained showed a whitish mycelium with a tan brown center that was in line with the original description of C. buxicola (2). DNA was extracted from the pure culture and the internal transcribed spacer (ITS) region was amplified by PCR using the ITS1-ITS4 primer pair (4). Nucleotide sequence was determined and deposited on GenBank (Accession No. JQ743502). BLAST analysis of the sequence showed 100% identity with all currently available C. buxicola ITS sequences, which confirmed our morphological diagnosis. To our knowledge, this is the first report of C. buxicola (teleomorph Calonectria pseudonaviculata) in France. The occurrence of this disease in France worries the nursery industry since losses can sometimes be dramatic as seen in United Kingdom, where the disease is widespread (3). References: (1) B. Henricot. The Plantsman 9:153, 2006. (2) B. Henricot and A. Culham. Mycologia 94:980, 2002. (3) B. Henricot et al. Plant Pathol. 49:805, 2000. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications, 1990.

Ceratocystis paradoxa (Dade) C. Moreau is a polyphagous wound parasite causing black rot post-harvest disease in pineapple. This fungus is responsible for high losses of fruit destined for the fresh market and is a common problem in many countries (2). C. paradoxa is officially listed as a quarantine pathogen for French Guiana. In November 2013, the Plant Protection Service of French Guiana observed damage in crops of Ananas comosus var. perolera located in Corossony (4°19′10.8″ N, 52°10′17.1″ W) and Wayabo (5°01′02.3″ N, 52°36′18.7″ W) (eastern and middle part of the country). The three plants collected at each location showed a soft base rot of the stem and of young leaves, and developed a foul smell. Plant tissues were collected from the edge of the lesions, chopped in small pieces, and plated on malt extract agar supplemented with 100 ppm chloramphenicol. The plates were incubated at 25°C with a 12-h photoperiod. After 5 days, a fungal colony, first white and downy, then becoming black and velvety after 10 days, was transferred on potato dextrose agar (PDA) and incubated in the same conditions. After 7 days, the colonies produced phialides releasing cylindrical or doliform conidia that were unicellular, colorless to pale brown, in long chains (3.09 to 20.17 × 3.1 to 5.57 μm, n = 20) and oval, pyriform, brown chlamydospores (8.02 to 21.32 × 4.20 to 9.76 μm, n = 20), occurring in long chains or singly with a vertical slit, usually not very visible. Furthermore, the colonies emitted a fruity odor. On the basis of these morphological characteristics, the fungus was identified as the anamorph of C. paradoxa (Thielaviopsis paradoxa (De Seynes) Höhn.) (1). The species designation was confirmed by sequencing the ITS region of the rDNA followed by comparison with reference sequences available in GenBank. Fungal material was collected from PDA culture by scraping the mycelium with a sterile needle and transferring into 2-ml microtubes. Fungal total DNA was then extracted and the ITS region was amplified by PCR using the ITS1-ITS4 primer pair. Nucleotide sequence was determined and deposited in GenBank (KJ667047). BLAST analysis showed 100% identity with C. paradoxa. The pathogenicity of the fungus was confirmed by inoculating two pineapples with mycelium from the C. paradoxa isolate grown on PDA. The peel of fruits and the base of the crowns were wounded with a sterile scalpel blade, each at five locations. Mycelial plugs (avg. 4 mm diameter) were placed on the wounds. Inoculation sites were wrapped with Parafilm to prevent dehydration and to hold the mycelial plugs in position. Negative controls received five sterile PDA plugs. The samples were incubated at 25°C in a moist chamber with a 12-h photoperiod. Eight days after inoculation, negative controls remained symptomless, whereas characteristic soft, watery, and black rot lesions developed on the base of all the crowns that were inoculated with C. paradoxa. The pathogen was successfully re-isolated from symptomatic tissues, fulfilling Koch's postulate. To our knowledge, this is the first report of C. paradoxa on A. comosus in French Guiana, and quarantine measures have been enforced to prevent the spread of this pathogen that might also cause severe losses on other susceptible plant species that are important for the local market (e.g., banana, coconut, sugar cane). Pineapple has become a major crop in French Guiana, and is now subjected to a more intensive monitoring, which may explains why this disease was discovered recently. References: (1) T. R. Nag Raj and W. B. Kendrick. A Monograph of Chalara and Allied Genera. Wilfrid Laurier University Press, Waterloo, Ontario, 1975. (2) R. C. Ploetz et al., eds. Compendium of Tropical Fruit Diseases. American Phytopathological Society, St. Paul, MN, 1994.

The presence of Fusarium temperatum in France was investigated by analyzing 40 Fusarium strains, previously isolated from infected maize ears in 2011 and formerly identified as Fusarium subglutinans based on morphological characteristics. In this study, 26 strains out of the 40 were identified as F. temperatum and 14 as F. subglutinans based on sequencing of the translation elongation factor 1α gene. The phylogenetic analysis showed that the two species represented two clades strongly supported by bootstrap values. The pathogenicity of F. temperatum strains was confirmed on maize ears. This study provides new information about F. temperatum isolated from maize in France.

Effective detection of pathogens from complex substrates is a challenging task. Molecular approaches such as real-time PCR can detect pathogens present even in low quantities. However, weak real-time PCR signals, as represented by high cycle threshold (Ct) values, may be questionable. Therefore, setting a reliable Ct threshold to declare a positive reaction is important for specific detection. In this study, five methods were assessed for their performance in determining a Ct cut-off value. These methods were based on the widely used probability of detection (POD) or receiver-operating characteristic (ROC) approaches. Two important forest pathogens, Hymenoscyphus fraxineus and Fusarium circinatum, were used to set up three experimental frameworks that combined two types of substrates (seed lots and spore traps) and different PCR machines. The ROC-based method emerged as the most complete and flexible method under various experimental conditions. It was demonstrated that the ROC method leads to a cut-off value below which late Ct results can reliably be considered indicative of positive test results. This cut-off value must be determined for each experimental approach used. The method based on the distribution of a previously determined set of Ct values corresponding to false-positives appeared to be better adapted to detecting false-negative results, and thus useful for testing potentially invasive pathogens.

Phytophthora lateralis is an oomycete responsible for Port-Orford Cedar (Chamaecyparis lawsoniana) dieback and mortality since the 1920s in western North America. It is recommended for quarantine regulation by EPPO (A2 list) and has been recently detected in Europe. In order to implement efficient control of the disease, a sensitive real-time PCR assay was developed to detect the presence of P. lateralis in plant tissues. In this study, we used the species-specific polymorphisms within the RAS-related protein gene Ypt1 to design a primer pair and a hydrolysis probe targeting the pathogen. The tool proved to be specific and inclusive, based on in silico and in vitro assessments, and could detect as few as 47 copies of target DNA. In addition, the test demonstrated its robustness and remained highly sensitive and specific under deliberately modified experimental conditions such as the hybridization temperature, or the reaction and DNA template volumes. The DNA extraction step from diseased tissues was also optimized, and the reliability of the results was ensured by a set of controls and a test targeting plant DNA that enabled the assessment of the quality of the extracts.

Fusarium oxysporum f. sp. cubense (Foc) is a fungus causing Fusarium wilt of banana (Musa spp.). The fungus is divided into three races and 24 vegetative compatibility groups (VCG) of which VCG 01213/16, commonly known as Foc tropical race 4 (Foc TR4), is of particular concern. Foc TR4 severely affects Cavendish (AAA) bananas, which comprise about 50% of all bananas produced globally, as well as many varieties susceptible to the other races of Foc. The pathogen was restricted to Southeast Asia and Australia until 2012, where after it has been detected in the Middle East, Mozambique in Africa, and Colombia in South America (Viljoen et al. 2020). Here we report the first detection of Foc TR4 in the French department of Mayotte, located in the Indian Ocean. In September 2019, leaf yellowing and wilting symptoms were observed in individual plants of the banana subgroups Silk (AAB) (cv. "Kissoukari") and Bluggoe (ABB) (cv. "Baraboufaka"). The symptomatic individuals were found in private gardens in the village of Poroani in Southwest Mayotte (World Geodetic System [WGS] 12° 53' 31.83''S, 45° 8' 30.98" E). When the pseudostems of symptomatic plants were split open, dark red to brown vascular discoloration was observed. Pseudostem tissue samples were collected and identified as Foc TR4 with the real-time PCR assay developed by Aguayo et al. (2017). Sections of the pseudostem samples were surface sterilized and used to isolate the fungus on potato dextrose agar (PDA) medium. Isolates were identified as F. oxysporum based on cultural and morphological characteristics as described in Leslie and Summerell (2006), which included fluffy aerial mycelia on PDA and the presence of short monophialides conidigenous cells bearing microconidia arranged in false heads. Abundant chlamydospores were also produced on synthetic nutrient poor agar (SNA) media. Single-spored isolates were used to develop nit mutants for vegetative compatibility group (VCG) testing (Correll 1991; Puhalla 1985). The isolates were confirmed as VCG 01213/16 as formation of heterokaryons was obtained with the nit mutants of the universal Foc TR4 tester. Two VCG 01213/16 isolates were then selected for pathogenicity testing by inoculating 2-month-old tissue culture-derived Cavendish plants, using the method described by Viljoen et al. (2017). After 10 weeks, the Foc TR4-inoculated plants produced wilting symptoms and internal rhizome discoloration typical of Fusarium wilt. Fusarium oxysporum was re-isolated from the inoculated plants and identified as Foc TR4/VCG 01213/16 by PCR (Dita et al. 2010; Matthews et al. 2020), thereby fulfilling Koch's postulates. Local authorities have destroyed the infected plants, and have undertaken an extensive survey to determine the distribution of Foc TR4 on the island. Three additional positive cases, identified with the real-time PCR assay of Aguayo et al. (2017), were found in the localities of Koungou ([WGS] 12° 44' 03''S, 45° 12' 08" E) and Boueni ([WGS] 12° 54' 25''S, 45° 04' 43" E). These included infected Cavendish banana (AAA) plants (cv. "Kontrike"). This is the first time that Foc TR4 has been found on a banana variety other than Cavendish when newly detected in a country. Considering the proximity of Mayotte to other islands of the Comoros archipelago, Madagascar and the East African coast, where banana is considered an important staple, this report describes a serious threat to banana production and the livelihoods of people in the region.

Brown rot is an economically important fungal disease affecting stone and pome fruit orchards, as well as harvested fruit during storage and on the market. Monilinia fructicola, M. laxa, and M. fructigena are the main causal agents of this disease and each have a different regulatory status depending on regional regulations. In this study, a new multiplex tool based on real-time polymerase chain reaction was developed to detect the three pathogenic fungi in a single reaction on fruit, twigs, and flowers of Prunus and Malus spp. Species-specific primer-hydrolysis probe combinations were designed to amplify a region located in a previously described MO368 sequenced characterized amplified region marker, and used in a quadruplex format coupled with the 18S Uni universal primer-probe test in order to check the quality of the DNA template. The assay was designed and optimized with the objective to provide high performance values. Experimental data supported its sensitivity, specificity, reproducibility, and robustness. In addition, a set of quality controls was implemented to minimize the risk of false-positive and false-negative results, thus making this new test fit for use in serial analyses and reliable in the framework of official controls.

Producción Científica, Fusarium circinatum Nirenberg & O’Donnel is listed among the species recommended for regulation as quarantine pests in Europe. Over 60 Pinus species are susceptible to the pathogen and it also causes disease on Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and species in genera such as Picea and Larix. The European Food Safety Authority considers the probability of new introductions—via contaminated seeds, wood material, soil and growing substrates, natural means and human activities—into the EU very likely. Due to early detection, constant surveillance and control measures, F. circinatum outbreaks have officially been eradicated in Italy and France. However, the global spread of F. circinatum suggests that the pathogen will continue to be encountered in new environments in the future. Therefore, continuous surveillance of reproductive material, nurseries and plantations, prompt control measures and realistic contingency plans will be important in Europe and elsewhere to limit disease spread and the “bridgehead effect”, where new introductions of a tree pathogen become increasingly likely as new environments are invaded, must be considered. Therefore, survey programs already implemented to limit the spread in Europe and that could be helpful for other EU countries are summarized in this review. These surveys include not only countries where pitch canker is present, such as Portugal and Spain, but also several other EU countries where F. circinatum is not present. Sampling protocols for seeds, seedlings, twigs, branches, shoots, soil samples, spore traps and insects from different studies are collated and compiled in this review. Likewise, methodology for morphological and molecular identification is herein presented. These include conventional PCR with a target-specific region located in the intergenic spacer region, as well as several real-time PCR protocols, with different levels of specificity and sensitivity. Finally, the global situation and future perspectives are addressed., European Cooperation in Science and Technology (COST Action FP1406 PINESTRENGTH), Ministerio de Economía, Industria y Competitividad - Fondo Europeo de Desarrollo Regional (project AGL2015-69370-R), Portuguese Foundation for Science and Technology - Fondo Europeo de Desarrollo Regional (project POCI-01-0145-FEDER-016785), Portuguese Foundation for Science and Technology (project PTDC/AGR-FOR/2768/2014 and grant SFRH/BPD/122928/2016), Centro de Estudios do Ambiente e do Mar (CESAM) (project UID/AMB/50017/2019)

International audience; Four species of the destructive forest pathogen Heterobasidion annosum sensu lato (s.l.) are present in Europe: H. annosum sensu stricto (s.s.), H. abietinum and H. parviporum are native species, while H. irregulare is a non-native invasive species currently reported only in Italy, yet recommended for regulation throughout Europe. In this study, real-time PCR detection tests were developed for each of the four species, which can be used simultaneously or individually thanks to probes labelled with species-specific fluorescent dyes. The different performance criteria of each assay were evaluated, and it was determined that they were theoretically capable of detecting amounts of DNA corresponding to 311, 29 and 29 cell nuclei in H. annosum s.s., H. irregulare and H. parviporum, respectively. The specificity of each assay was assessed with a wide set of strains. Real-time PCR tests successfully detected Heterobasidion species from 36 fruiting bodies taken from the forest, as well as from artificially inoculated or naturally infected wood samples. The multiplex real-time PCR assays developed in this study could have practical applications both in forest management and in phytosanitary monitoring.

Producción Científica, Fusarium circinatum (Nirenberg and O’ Donnell) is the causal agent of pine pitch canker (PPC) disease, one of the most devastating forest diseases worldwide. Long-distance spread occurs mainly through the movement of infected seeds whereas at regional level, the movement of seedlings, substrates, or containers may play an important role in fungal dispersal. Invasion of nurseries takes place via infected seeds and further spread can occur by planting contaminated seedlings, especially due to the possibility of infected plants remaining symptomless. Once established, F. circinatum spreads by rain, wind, and insects. The natural spread of the pathogen is limited due to the short dispersal distances of the spores and the fairly short flight distances of disseminating insects. In this review, we summarize the currently known dispersal pathways of the pathogen, discussing both natural and human-assisted processes. With the purpose of understanding how to best intervene in the disease’s development in nurseries and forests, we outline the epidemiology of the pathogen describing the key factors influencing its spread. Preventive measures to control the spread of F. circinatum locally and globally are described with special emphasis on the challenges in implementing them., European Cooperation in Science and Technology (COST Action FP1406 PINESTRENGTH), Ministerio de Economía, Industria y Competitividad - Fondo Europeo de Desarrollo Regional (project AGL2015-69370-R), Centro de Estudios do Ambiente e do Mar (CESAM) (project UID/AMB/50017/2019), Portuguese Foundation for Science and Technology (projects PTDC/AGR-FOR/2768/2014 , SFRH/BPD/122928/2016), French National Research Agency (grant ANR-11-LABX-0002-01)

Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis are causal agents of citrus scab and spot diseases. The three pathogens are listed as quarantine pests in many countries and are subject to phytosanitary measures to prevent their entry. Diagnosis of these diseases based on visual symptoms is problematic, as they could be confused with other citrus diseases. Isolation of E. fawcettii, E. australis, and P. angolensis from infected tissues is challenging because they grow slowly on culture media. This study developed rapid and specific detection tools for the in planta detection of these pathogens, using either conventional PCR or one-tube multiplex real-time PCR. Primers and hybridization probes were designed to target the single-copy protein-coding gene MS204 for E. fawcettii and E. australis and the translation elongation factor (Tef-1α) gene for P. angolensis. The specificity of the assays was evaluated by testing against DNA extracted from a large number of isolates (102) collected from different citrus-growing areas in the world and from other hosts. The newly described species E. citricola was not included in the specificity test due to its unavailability from the CBS collection. The detection limits of conventional PCR for the three pathogens were 100, 100, and 10 pg μl−1 gDNA per reaction for E. fawcettii, E. australis, and P. angolensis, respectively. The quadruplex qPCR was fully validated assessing the following performance criteria: sensitivity, specificity, repeatability, reproducibility, and robustness. The quadruplex real-time PCR proved to be highly sensitive, detecting as low as 243, 241, and 242 plasmidic copies (pc) μl−1 of E. fawcettii, E. australis, and P. angolensis, respectively. Sensitivity and specificity of this quadruplex assay were further confirmed using 176 naturally infected citrus samples collected from Ethiopia, Cameroon, the United States, and Australia. The quadruplex assay developed in this study is robust, cost-effective, and capable of high-throughput detection of the three targets directly from citrus samples. This new detection tool will substantially reduce the turnaround time for reliable species identification and allow rapid response and appropriate action.

International audience; Phytophthora ramorum Werres, De Cock & Man in’t Veld, an oomycete known in the United States as the causal agent of sudden oak death, has spread across Europe since the early 2000s. It is responsible for damage and death to a wide range of plant species, including mature trees. In 2009 it was identified on Japanese larch (Larix kaempferi) in southwest England (Webber et al. 2010), and since then it has caused severe damage and losses to Larix spp. in the United Kingdom and the Republic of Ireland. There are two lineages of the oomycete, EU1 and EU2, found in Europe (King et al. 2015), EU2 being the more aggressive. The symptoms on larch include necrosis and loss of needles, wilting of shoots, dieback of branches, and death, often with abundant resin bleeding on trunks and branches. As sporulating hosts, Larix spp. may disperse P. ramorum over long distances. In May 2017, wilting, yellowing/reddening needles, and branch mortality were observed on mature Larix kaempferi (about 50 years old) in the forest of Saint-Cadou, Finistère, in the far northwestern part of France (3°59′49.2″ W; 48°22′22.4″ N). Approximately, 27% of the trees were affected in May, and 42% later in September 2017. The presence of P. ramorum was suspected and was first confirmed by testing samples collected from trunks and branches with necrosis and resin bleeds, using the specific conventional polymerase chain reaction (PCR) method developed by Ioos et al. (2006). The oomycete was also isolated in pure culture, using a Phytophthora selective medium (PARB[H]). The features observed, such as a coralloid mycelium, the presence of numerous thin-walled chlamydospores (up to 75 µm large), and deciduous, semipapillate sporangia arranged in clusters, matched those reported for P. ramorum. In June 2017, the presence of P. ramorum was confirmed in another larch stand in Hanvec, Finistère (4°12′45.0″ W; 48°20′10.8″ N), using the same identification techniques. In this stand, the prevalence was not precisely estimated but was deemed much lower than in Saint-Cadou. Based on the analysis of Cox1 partial sequence and the PCR restriction fragment length polymorphism pattern described by Van Poucke et al. (2012) on Cox1, the P. ramorum isolates collected in these two forests could be assigned to the EU1 lineage. This is the first report of P. ramorum affecting Japanese larch in France and in mainland Europe. Until now it had only been detected on shrubs in nurseries, green spaces, and in rare circumstances in the natural environment on understory vegetation (rhododendron) in Normandy and Brittany, but not in the vicinity of the infected larch stands. The presence of this pathogen in the natural environment represents a major threat for larch trees, but also for the other potential forest host trees in this region, such as sweet chestnut, and might have a severe impact on both forest and ornamental tree species. Research is in progress to learn more about this outbreak, the possible origin of the inoculum, the extension of the disease, and its progression.

International audience; Ash dieback is caused by an invasive pathogen, Hymenoscyphus fraxineus, which emerged in Europe in the 1990s and jeopardizes the management of ash stands. Although the biological cycle of the pathogen is well understood, its dispersal patterns via airborne spores remain poorly described. We investigated the seasonal and spatial patterns of dispersal in France using both a passive spore-trapping method coupled with a real-time PCR assay and reports of ash dieback based on symptom observations. Spores detection varies from year to year, with a detection ability of 30 to 47%, depending on meteorological conditions, which affect both production of inoculum and efficiency of the trapping. Nevertheless, our results are consistent and we showed that sporulation peak occurred from June to August and that spores were detected up to 50-100 km ahead of the disease front, proving the presence of the pathogen before any observation of symptoms. The spore dispersal gradient was steep, most of inoculum remaining within 50 m of infected ashes. Two dispersal kernels were fitted using Bayesian methods to estimate the mean dispersal distance of H. fraxineus from inoculum sources. The estimated mean distances of dispersal, either local or regional scale, were 1.4 km and 2.6 km, respectively, the best fitting kernel being the inverse power-law. This information may help to design disease management strategies.

Techniques based on high-throughput sequencing (HTS) of environmental DNA have provided a new way of studying fungal diversity. However, these techniques suffer from a number of methodological biases which may appear at any of the steps involved in a metabarcoding study. Air is one of the most important environments where fungi can be found, because it is the primary medium of dispersal for many species. Looking ahead to future developments, it was decided to test 20 protocols, including different passive spore traps, spore recovery procedures, DNA extraction kits, and barcode loci. HTS was performed with the Illumina MiSeq platform targeting two subloci of the fungal internal transcribed spacer. Multivariate analysis and generalized linear models showed that the type of passive spore trap, the spore recovery procedure, and the barcode all impact the description of fungal communities in terms of richness and diversity when assessed by HTS metabarcoding. In contrast, DNA extraction kits did not significantly impact these results. Although passive traps may be used to describe airborne fungal communities, a study using specific real-time PCR and a mock community showed that these kinds of traps are affected by environmental conditions that may induce losses of biological material, impacting diversity and community composition results. IMPORTANCE The advent of high-throughput sequencing (HTS) methods, such as those offered by next-generation sequencing (NGS) techniques, has opened a new era in the study of fungal diversity in different environmental substrates. In this study, we show that an assessment of the diversity of airborne fungal communities can reliably be achieved by the use of simple and robust passive spore traps. However, a comparison of sample processing protocols showed that several methodological biases may impact the results of fungal diversity when assessed by metabarcoding. Our data suggest that identifying these biases is of paramount importance to enable a correct identification and relative quantification of community members.

Fusarium circinatum and Caliciopsis pinea are the causal agents of Pitch canker and Caliciopsis canker, respectively. These diseases affect pines and other conifers both in Europe and North America. The two pathogens cause similar bleeding cankers, especially at the early stage of colonization. Symptoms closely resembling those due to F. c i r c i n at um can be instead associated with C. pinea.SinceF. c i rc i n at um is a quarantine organism, subjected to provisional emergency measures, its report immediately causes serious economic implications, while C. pinea, even if now emerging, is not regulated in the EU nor in the USA. For this reason, a reliable and accurate diagnostic tool able to distinguish between the two organisms was considered a priority. In this study, we developed and standardized a duplex real-time PCR assay allowing the simultaneous recognition of C. pinea and F. circinatum DNA in pine tissue in a reasonably short time and for amounts as small as 0.06 pg/?l. The molecular assay is, therefore, able to detect the infection even before symptoms have fully developed. The test was challenged with a very large set of strains (110 different isolates) collected in different regions of the world and host trees, and gave reliable results. The high efficiency of this method suggests its use as a standard diagnostic tool during phytosanitary controls. In addition, the duplex realtime PCR assay presented here is the first DNA-based method designed to detect C. pinea, which is becoming an increasing threat to pine stands both in North America and in Europe.

Accelerating international trade and climate change make pathogen spread an increasing concern. Hymenoscyphus fraxineus, the causal agent of ash dieback, is a fungal pathogen that has been moving across continents and hosts from Asian to European ash. Most European common ash trees (Fraxinus excelsior) are highly susceptible to H. fraxineus, although a minority (~5%) have partial resistance to dieback. Here, we assemble and annotate a H. fraxineus draft genome, which approaches chromosome scale. Pathogen genetic diversity across Europe and in Japan, reveals a strong bottleneck in Europe, though a signal of adaptive diversity remains in key host interaction genes. We find that the European population was founded by two divergent haploid individuals. Divergence between these haplotypes represents the ancestral polymorphism within a large source population. Subsequent introduction from this source would greatly increase adaptive potential of the pathogen. Thus, further introgression of H. fraxineus into Europe represents a potential threat and Europe-wide biological security measures are needed to manage this disease.

Accelerating international trade and climate change make pathogen spread an increasing concern. Hymenoscyphus fraxineus, the causal agent of ash dieback is one such pathogen, moving across continents and hosts from Asian to European ash. Most European common ash (Fraxinus excelsior) trees are highly susceptible to H. fraxineus although a small minority (~5%) evidently have partial resistance to dieback. We have assembled and annotated a draft of the H. fraxineus genome which approaches chromosome scale. Pathogen genetic diversity across Europe, and in Japan, reveals a tight bottleneck into Europe, though a signal of adaptive diversity remains in key host interaction genes (effectors). We find that the European population was founded by two divergent haploid individuals. Divergence between these haplotypes represents the 'shadow' of a large source population and subsequent introduction would greatly increase adaptive potential and the pathogen's threat. Thus, EU wide biological security measures remain an important part of the strategy to manage this disease.

Citrus products are economically important in Tunisia and constitute a significant part of agricultural exports. In March and April 2019, symptoms resembling citrus black spot were observed on citrus fruit (Citrus limon and C. sinensis) in …

Fusarium graminearum is an important pathogen causing Fusarium head blight (FHB) on wheat and barley and Fusarium ear rot (FER) on maize, and harvested grains often are contaminated with trichothecenes such as deoxynivalenol (DON) and nivalenol (NIV) that are a major health and food safety concern due to their toxicity to humans and farm animals. In this study, species identity and trichothecene toxin potential of 294 members of the Fusarium graminearum species complex (FGSC) collected from wheat, barley and maize in France in 2011 was determined using a microsphere-based multilocus genotyping assay. F. graminearum was predominant on all three hosts, but three isolates of F. cortaderiae and two isolates representing F. graminearum × F. boothii hybrids were also identified from maize. The 15-ADON trichothecene chemotype predominated on all three hosts, representing 94.7 %, 87.8 % and 85.4 % of the strains on barley (N = 19), wheat (N = 90), and maize (N = 185), respectively. However, the NIV chemotype was found in 12.2 % of the wheat isolates and in 14.6 % of the maize isolates. Only a single FGSC isolate from this study, originating from barley, was found to have the 3-ADON chemotype. Regional differences could be observed in the distribution of the 15-ADON and NIV chemotypes, with the NIV producing-isolates being present at higher frequency (21.2 %) in the South of France compared to the rest of the country (4.4 %). Such information is critical because of the increased concern associated with NIV contamination of cereals. In addition, these results are needed to develop management strategies for FHB and FER in France and to improve understanding of the distribution and significance of FGSC diversity in Europe and worldwide.

Gibberella circinata (anamorph Fusarium circinatum) is a harmful fungus infecting pine trees that has been subject to European Union emergency phytosanitary measures since 2007. This fungus is spread over long distances primarily via pine seeds, consequently reliable detection tools and methods are needed to test pine seed lots imported and marketed in the EU. Several detection methods are available in the scientific literature but very limited validation data is available. For that purpose, one European EUPHRESCO project called ‘Gibcir-Diagseed’ was launched, involving 10 countries. This project aimed to select and validate one or more diagnostic procedures targeting this pest via inter-laboratory tests, as well as a suitable sampling method. Performance criteria were calculated for three procedures chosen by participants: isolation followed by morphological characterization, isolation followed by conventional PCR and biological enrichment followed by real-time PCR. The results showed that all three procedures provided acceptable performance criteria but also demonstrated that none of them could generate 100% of correct results. Regarding the sampling issue, the National Plant Protection Organizations consultation enabled an agreement to be reached on the number of seeds to be tested: 400 per sample, for a 95% confidence level that an infection of at least 1% will be detected. Etude de performance des procedures de diagnostic pour l'identification et la detection de Gibberella circinata dans les semences de pin dans le cadre d'un projet EUPHRESCO Gibberella circinata (anamorphe Fusarium circinatum) est un champignon pathogene infectant les pins qui fait l'objet de mesures phytosanitaires d'urgence au sein des pays de l'Union europeenne depuis 2007. Ce champignon est dissemine sur des distances importantes essentiellement par le biais de semences de pin. Par consequent, des outils et des methodes de detection fiables sont necessaires pour tester les lots de semences de pin importes et commercialises dans l'Union europeenne. Plusieurs methodes de detection sont decrites dans la litterature scientifique mais il y a peu de donnees de validation disponibles. A cet effet, un projet europeen EUPHRESCO nomme « Gibcir-Diagseed » a ete lance en impliquant 10 pays. Ce projet vise a selectionner et valider une ou plusieurs procedures ciblant cet organisme au travers de tests inter-laboratoires, en meme temps qu'une methode d’echantillonnage adaptee. Des criteres de performance ont ete calcules pour trois procedures de diagnostic choisies par les participants: isolement suivi de la caracterisation morphologique, isolement suivi d'une PCR conventionnelle, et enrichissement biologique suivi par une PCR en temps reel. Les resultats ont montre que les trois procedures avaient des criteres de performance acceptables mais ont egalement demontre qu'aucune d'entre elles ne pouvait generer 100% de resultats corrects. Au sujet de l’echantillonnage, la consultation des Organisations Nationales de la Protection des Vegetaux a permis d'atteindre un consensus sur le nombre de semences a tester: 400 par echantillon, pour detecter un niveau d'infection d'au moins 1% avec un niveau de confiance de 95%. Иccлeдoвaниe кaчecтвa тecтиpoвaния диaгнocтичecкиx пpoцeдyp пo идeнтификaции и выявлeнию Gibberella circinata в ceмeнax cocны в paмкax пpoeктa EUPHRESCO Gibberella circinata (aнaмopфнaя cтaдия Fusarium circinatum) являeтcя вpeдным гpибoм, зapaжaющим cocнy, кoтopый являeтcя oбъeктoм чpeзвычaйныx фитocaнитapныx мep Eвpoпeйcкoгo Coюзa (EC) c 2007 г. Этoт гpиб pacпpocтpaняeтcя нa бoльшиe paccтoяния, пpeждe вceгo c ceмeнaми cocны, и пoэтoмy нeoбxoдимы нaдeжныe cpeдcтвa oбнapyжeния и мeтoды, пoзвoляющиe пpoвepять пapтии ceмян cocны, кoтopыe импopтиpyютcя и пpoдaютcя в EC. B нayчнoй литepaтype дaeтcя oпиcaниe нecкoлькиx мeтoдoв oбнapyжeния, oднaкo cyщecтвyют лишь oгpaничeнныe дaнныe o вaлидaции. C этoй цeлью был пpeдпpинят пpoeкт EC EUPHRESCO пoд нaзвaниeм “Gibcir-Diagseed”, в кoтopый былo вoвлeчeнo 10 cтpaн. Этoт пpoeкт нaцeлeн нa oтбop и вaлидaцию oднoй или бoлee пpoцeдyp для идeнтификaции этoгo вpeднoгo opгaнизмa пyтeм тecтиpoвaния в нecкoлькиx лaбopaтopияx, a тaкжe пoдxoдящий мeтoд oтбopa oбpaзцoв. Кpитepии кaчecтвa aнaлизa были пoдcчитaны для тpex пpoцeдyp, выбpaнныx yчacтникaми: выдeлeниe c пocлeдyющeй xapaктepизaциeй мopфoлoгии, изoляция c пocлeдyющим oбычным ПЦP и биoлoгичecкoe oбoгaщeниe c пocлeдyющим ПЦP в peaльнoм вpeмeни. Peзyльтaты пoкaзaли, чтo вce тpи пpoцeдypы oбecпeчили пpиeмлeмыe кpитepии кaчecтвa, нo тaкжe пpoдeмoнcтpиpoвaли, чтo ни oдин из ниx нe мoг дaть 100%-yю пpaвильнocть peзyльтaтoв. B oтнoшeнии oтбopa oбpaзцoв, кoнcyльтaция c нaциoнaльными opгaнизaциями пo кapaнтинy и зaщитe pacтeний пoзвoлилa бы пpийти к coглaшeнию пo кoличecтвy тecтиpyeмыx ceмян: 400 в кaждoм oбpaзцe, для 95%-гo дoвepитeльнoгo ypoвня, пpи кoтopoм бyдeт выявлятьcя, пo кpaйнeй мepe, 1%-нaя зapaжeннocть.