Simulation of consumer exposure to deoxynivalenol according to wheat crop management and grain segregation: Case studies and methodological considerations

International audienceWe combined agronomic data and a model simulating exposure based on consumption data to assess the impact of crop management and grain segregation procedures on consumer exposure to deoxynivalenol. We used three scenarios of soil tillage at a regional scale and three scenarios of grain segregation for a supply area. The soil tillage scenarios were applied to a range of mean crop contamination levels, with various coefficients representing the degree of tillage. The grain segregation scenarios were applied to two real datasets of DON content distributions. We found that the increase in consumer exposure in response to increases in "risky" crop management practices such as direct-drilling depends largely on mean contamination and on the value of the tillage coefficient. The results for grain segregation procedures showed that exposure was most strongly affected by contamination distributions as the segregation procedure minimising risk differed for the two datasets

Interlaboratory performance of a Real-Time PCR method for detection of Ceratocystis platani, the agent of canker stain of Platanus spp

Ceratocystis platani (CP), an ascomycetous fungus, is the agent of canker stain, a lethal vascular disease of Platanus species. Ceratocystis platani has been listed as a quarantine pest (EPPO A2 list) due to extensive damage caused in Southern Europe and the Mediterranean region. As traditional diagnostic assays are ineffective, a Real-Time PCR detection method based on EvaGreen, SYBR Green, and Taqman assays was previously developed, validated in-house, and included in the official EPPO standard PM7/14 (2). Here, we describe the results of a test performance study performed by nine European laboratories for the purpose of an interlaboratory validation. Verification of the DNA extracted from biological samples guaranteed the high quality of preparations, and the stability and the homogeneity of the aliquots intended for the laboratories. All of the laboratories reproduced nearly identical standard curves with efficiencies close to 100%. Testing of blind-coded DNA extracted from wood samples revealed that all performance parameters-diagnostic sensitivity, diagnostic specificity, accuracy and reproducibility-were best fit in most cases both at the laboratory and at the assay level. The previously established limit of detection, 3 fg per PCR reaction, was also validated with similar excellent results. The high interlaboratory performance of this Real-Time PCR method confirms its value as a primary tool to safeguard C. platani-free countries by way of an accurate monitoring, and to investigate the resistance level of potentially canker stain-resistant Platanus genotypes

Identification and pathogenicity of Alternaria species associated with leaf blotch disease and premature defoliation in French apple orchards

Leaf blotch caused by Alternaria spp. is a common disease in apple-producing regions. The disease is usually associated with one phylogenetic species and one species complex, Alternaria alternata and the Alternaria arborescens species complex (A. arborescens SC), respectively. Both taxa may include the Alternaria apple pathotype, a quarantine or regulated pathogen in several countries. The apple pathotype is characterized by the production of a host-selective toxin (HST) which is involved in pathogenicity towards the apple. A cluster of genes located on conditionally dispensable chromosomes (CDCs) is involved in the production of this HST (namely AMT in the case of the apple pathotype). Since 2016, leaf blotch and premature tree defoliation attributed to Alternaria spp. have been observed in apple-producing regions of central and south-eastern France. Our study aimed to identify the Alternaria species involved in apple tree defoliation and assess the presence of the apple pathotype in French orchards. From 2016 to 2018, 166 isolates were collected and identified by multi-locus sequence typing (MLST). This analysis revealed that all these French isolates belonged to either the A. arborescens SC or A. alternata. Specific PCR detection targeting three genes located on the CDC did not indicate the presence of the apple pathotype in France. Pathogenicity was assessed under laboratory conditions on detached leaves of Golden Delicious and Gala apple cultivars for a representative subset of 28 Alternaria isolates. All the tested isolates were pathogenic on detached leaves of cultivars Golden Delicious and Gala, but no differences were observed between the pathogenicity levels of A. arborescens SC and A. alternata. However, the results of our pathogenicity test suggest that cultivar Golden Delicious is more susceptible than Gala to Alternaria leaf blotch. Implications in the detection of the Alternaria apple pathotype and the taxonomic assignment of Alternaria isolates involved in Alternaria leaf blotch are discussed

Global Geographic Distribution and Host Range of Fusarium circinatum, the Causal Agent of Pine Pitch Canker

Funding: This study was financially supported by 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. ANSES is supported by a grant managed by the French National Research Agency (ANR) as part of the “Investissements d’Avenir” programme (ANR-11-LABX-0002-01, Laboratory of ExcellenceARBRE). SW was partly supported by BBSRC Grant reference BB/L012251/1 “Promoting resilience of UK tree species to novel pests & pathogens: ecological & evolutionary solutions (PROTREE)” jointly funded by BBSRC, Defra, ESRC, the Forestry Commission, NERC and the Scottish Government, under the Tree Health and Plant Biosecurity Initiative. Annual surveys in Switzerland were financially supported by the Swiss Federal Office for the Environment FOEN. Acknowledgments: Andrea Kunova and Cristina Pizzatti are acknowledged for the assistance in the sampling. Thanks are due to Dina Ribeiro and Helena Marques from ICNF-Portuguese Forest Authority for providing location coordinates. We thank three anonymous reviwers for valuable corrections and suggestions.Peer reviewedPublisher PD

Caractérisation génétique de Phytophthora alni Brasier & S.A. Kirk, hybride interspécifique agent du dépérissement de l'aulne en Europe

An emergent disease of alder is caused by a complex of three taxa belonging to the genus Phytophthora (Oomycetes): P. alni subsp. alni (Paa), P. alni subsp. multiformis (Pam) and P. alni subsp. uniformis (Pau). The first part of this study focused on the development of specific detection tools for these three taxa. Based on SCARs generated with RAPD, we designed three PCR primer pairs which can be combined to specifically detect and identify Paa, Pam and Pau in different substrates (plant tissue, water, soil). Second, we studied the occurrence and the allelic distribution for several nuclear single-copy genes containing introns on a wide collection of P. alni and close species. Mitochondrial DNA was also studied through RFLP and gene sequencing. We demonstrated that i) Pau may not result from a hybridization event, ii) two divergent alleles for each of the nuclear genes are observed in Pam, which suggests this taxon may have been generated by a reticulation or by autopolyploidisation, iii) Paa combines the alleles observed in Pam and Pau and was probably generated by hybridization between Pam and Pau or Pam- and Pau-like taxa. In addition, we studied the expression of elicitin genes, a multigenic family specific to the genus Phytophthora. The cumulative patterns of Pau and Pam in regard with Paa confirmed our first results. Last, in order to study the genetic variability of the different taxa, microsatellite markers were isolated in Paa and characterized. The genotypes we resolved demonstrate a low level of variability for the three taxa. They confirm our hypotheses in regard with Paa origin and suggest that Pam is also an allopolyploid taxon.Une maladie émergente causant le dépérissement de l'aulne est causée par un complexe de trois taxons du genre Phytophthora (Oomycète) : P. alni subsp. alni (Paa), P. alni subsp. multiformis (Pam) et P. alni subsp. uniformis (Pau). La première partie de cette étude a consisté à mettre au point des outils de détection spécifiques de ces trois taxons. à partir de SCARs générés par RAPD, nous avons défini trois couples d'amorces de PCR dont l'utilisation combinée permet la détection et l'identification spécifique de Paa, Pam et Pau dans différents substrats (plante, eau, sol). Nous avons ensuite étudié la présence et la distribution allélique pour quatre gènes nucléaires contenant des introns sur une collection de P. alni et d'espèces proches. L'ADN mitochondrial a également été étudié par RFLP et séquençage de deux gènes. Nous avons montré que i) Pau ne semble pas avoir été généré par hybridation, ii) Pam présente deux allèles fortement divergents pour chaque gène nucléaire et résulterait donc d'une réticulation ou d'une autopolyploïdisation, iii) Paa cumule les allèles présents chez Pam et Pau et a probablement été créé par hybridation entre Pam et Pau ou des taxons très proches. De plus, nous avons étudié le profil d'expression des gènes codant pour des élicitines, famille multigénique spécifique du genre hytophthora. L'additivité des profils de Pau et Pam vis-à-vis de Paa a confirmé nos premiers résultats. Enfin, afin d'étudier la variabilité génétique de ces différents taxons, des marqueurs microsatellites ont été isolés chez Paa et caractérisés. Les génotypes obtenus montrent une faible variabilité chez les trois taxons. Ils confirment nos hypothèses quant à l'origine de Paa et suggèrent que Pam est aussi un taxon allopolyploïde

The Main Methods for Detecting Quarantine Pests (Abstract)

Dans le domaine forestier, la lutte contre les bioagresseurs de quarantaine commence par la prévention. Identifier rapidement et avec fiabilité un bioagresseur permet d’empêcher son entrée sur le territoire et d’envisager une éradication précoce en cas de découverte de foyer. Les méthodes d’identification utilisent principalement des techniques permettant de rechercher spécifiquement, a priori, un ou plusieurs bioagresseurs cibles dans un échantillon de plante ou de sol. L’essor des techniques d’amplification génique de type PCR (en point final ou en temps réel) a rendu ces détections très fiables et rapides. Une autre approche consiste à travailler sans a priori, en réalisant un inventaire microbiologique de l’échantillon à analyser. Elle a longtemps reposé sur des protocoles classiques de caractérisation morphologique, mais l’apport des nouvelles techniques de séquençage d’ADn [code-barres d’ADn (barcoding), séquençage environnemental (metabarcoding)] ou d’analyse du protéome (spectrométrie de masse MALDI-TOF) permet à présent d’identifier les bioagresseurs sans les rechercher a priori. Les perspectives en matière de méthode de détection sont nombreuses et s’orienteront peut-être vers des techniques ciblant plus spécifiquement les microorganismes sur la base de leur potentiel de pathogénicité.In the forestry area, quarantine pest control begins with prevention. Rapid, reliable identification of a pest helps to prevent it from entering the territory and to contemplate early eradication when an outbreak is discovered. Identification methods mainly call on techniques for previously specified target pests in a plant or soil sample. The development of PCR type techniques (end point or real time) has made these detections very fast and reliable. Another approach works without a pre-specified target by conducting a microbiological inventory of the test sample. For a long time this has relied on conventional protocols for morphological characterisation but new DnA sequencing techniques (DnA barcoding, meta-barcoding) or proteomic analysis (MALDI-TOF mass spectrometry) now allow identification of pests that are not pre-specified. This opens broad perspectives for detection methods which may evolve towards techniques that more specifically target microorganisms on the basis of their potential pathogenicity

First report of Dothistroma pini, a recent agent of the Dothistroma needle blight (DNB), on Pinus radiata in France

Dothistroma needle blight (DNB), also known as red band needle blight, is an important fungal disease of Pinus spp. that occurs worldwide. On the basis of molecular and morphological studies of the anamorphic stage, Barnes et al.(1) showed that two closely related species were involved in DNB: Dothistroma septosporum (Dorog.) Morelet and Dothistroma pini Hulbary. D. septosporum (teleomorph: Mycosphaerella pini Rostr.) has a worldwide distribution and is reported as the species that caused past epidemics of DNB. This species is reported on more than 80 different pine species, and P. radiata D. Don (Monterey pine) is classified as a highly or moderately susceptible species, depending on the published sources (4). D. pini (telemorph: unknown) was initially found on needles of P. nigra J. F. Arnold collected from 1964 to 2001 in North Central U.S.A. (Minnesota, Nebraska and Michigan). It was subsequently found in Ukraine and southwestern Russia, where it has been associated with the emergence of DNB on P. nigra subsp. pallasiana (Lamb.) Holmboe, in Hungary on P. nigra and in Russia on P. mugo Turra (1). In France, D. pini was reported for the first time on P. nigra, and was sometimes found in association with D. septosporum on the same needles (3). Later on, a more intensive survey of DNB was launched in France and 216 stands of Pinus sp. were studied. D. septosporum and D. pini were detected in 133 and 123 stands, respectively. Both species co-occurred in 40 stands but D. pini was only found on P. nigra (subsp. laricio and austriaca) (2). Up to now, D. pini was therefore only reported on European pine species, mainly on the different allopatric subspecies belonging to the black pine complex and on one occasion on P. mugo, which belongs to the same section and subsection as P. nigra. In March 2011, typical symptoms of DNB (needles with orangey-red brown distal ends, dark red bands and green bases; small and black fruit bodies within the bands) were observed in a 50 to 60-years old P. radiata stand, of approximately 3 ha., located in the Pyrénées Atlantiques, close to the Spanish border (Sare, 1° 36' 08'' West; 43° 19' 51'' North). The density of pine was relatively low and patches of natural regeneration were present. Although nearly all of the trees showed DNB symptoms, less than 50% of their needles were affected by the disease. In this stand, needles showing typical DNB symptoms were randomly taken from 4 pines and mixed together to form a single sample for analysis. Total DNA was extracted from symptomatic needle pieces. The presence of Dothistroma pini was confirmed by a specific multiplex real-time polymerase chain reaction (PCR) analysis using the D. pini-specific primers/probe combination DPtef-F1-/R1/-P1 (3), and by sequencing a D. pini-specific amplicon generated by another conventional PCR (3) using DPtef-F/DPtef-R primers (GenBank accession KC853059) (3). D. septosporum was not detected in the sample. To our knowledge, this is the first report worldwide of D. pini on P. radiata which is a pine species largely planted in Spain and in the Southern Hemisphere. This is also the first report of this pathogen on a non-European pine species. The original native range and the host range of D. pini remain unknown and there is currently no data about host preferences or aggressiveness on different pine species

Caractérisation génétique de Phytophthora alni Brasier & S.A. Kirk, hybride interspécifique agent du dépérissement de l'aulne en Europe

Une maladie émergente causant le dépérissement de l'aulne est causée par un complexe de trois taxons du genre Phytophthora (Oomycète) : P. alni subsp. alni (Paa), P. alni subsp. multiformis (Pam) et P. alni subsp. uniformis (Pau). La première partie de cette étude a consisté à mettre au point des outils de détection spécifiques de ces trois taxons. À partir de SCARs générés par RAPD, nous avons défini trois couples d'amorces de PCR dont l'utilisation combinée permet la détection et l'identification spécifique de Paa, Pam et Pau dans différents substrats (plante, eau, sol). Nous avons ensuite étudié la présence et la distribution allélique pour quatre gènes nucléaires contenant des introns sur une collection de P. alni et d'espèces proches. L'ADN mitochondrial a également été étudié par RFLP et séquençage de deux gènes. Nous avons montré que i) Pau ne semble pas avoir été généré par hybridation, ii) Pam présente deux allèles fortement divergents pour chaque gène nucléaire et résulterait donc d'une réticulation ou d'une autopolyploïdisation, iii) Paa cumule les allèles présents chez Pam et Pau et a probablement été créé par hybridation entre Pam et Pau ou des taxons très proches. De plus, nous avons étudié le profil d'expression des gènes codant pour des élicitines, famille multigénique spécifique du genre hytophthora. L'additivité des profils de Pau et Pam vis-à-vis de Paa a confirmé nos premiers résultats. Enfin, afin d'étudier la variabilité génétique de ces différents taxons, des marqueurs microsatellites ont été isolés chez Paa et caractérisés. Les génotypes obtenus montrent une faible variabilité chez les trois taxons. Ils confirment nos hypothèses quant à l'origine de Paa et suggèrent que Pam est aussi un taxon allopolyploïde.An emergent disease of alder is caused by a complex of three taxa belonging to the genus Phytophthora (Oomycetes): P. alni subsp. alni (Paa), P. alni subsp. multiformis (Pam) and P. alni subsp. uniformis (Pau). The first part of this study focused on the development of specific detection tools for these three taxa. Based on SCARs generated with RAPD, we designed three PCR primer pairs which can be combined to specifically detect and identify Paa, Pam and Pau in different substrates (plant tissue, water, soil). Second, we studied the occurrence and the allelic distribution for several nuclear single-copy genes containing introns on a wide collection of P. alni and close species. Mitochondrial DNA was also studied through RFLP and gene sequencing. We demonstrated that i) Pau may not result from a hybridization event, ii) two divergent alleles for each of the nuclear genes are observed in Pam, which suggests this taxon may have been generated by a reticulation or by autopolyploidisation, iii) Paa combines the alleles observed in Pam and Pau and was probably generated by hybridization between Pam and Pau or Pam- and Pau-like taxa. In addition, we studied the expression of elicitin genes, a multigenic family specific to the genus Phytophthora. The cumulative patterns of Pau and Pam in regard with Paa confirmed our first results. Last, in order to study the genetic variability of the different taxa, microsatellite markers were isolated in Paa and characterized. The genotypes we resolved demonstrate a low level of variability for the three taxa. They confirm our hypotheses in regard with Paa origin and suggest that Pam is also an allopolyploid taxon.NANCY1-SCD Sciences & Techniques (545782101) / SudocSudocFranceF

Development and use of new sensitive molecular tools for diagnosis and detection of Melampsora rusts on cultivated poplar

Poplar rusts due to Melampsora larici-populina (Mlp), M. allii-populina (Map) and M. medusae f. sp. deltoidae (Mmd) are the most serious disease in Europe on cultivated poplars, that is, Populus 9 euramericana and P. 9 interamericana hybrids. These pathogenic species can be identified by the observation of morphological characteristics of urediniospores but this method is not appropriate for high-throughput analysis and cannot be used on other spore stages, such as aeciospores or teliospores, that are morphologically similar. The aim of this study was to develop a rapid and sensitive molecular method based on PCR amplification that was able to specifically detect these species on various hosts for routine analysis. Three primer pairs ITS-MLP-F/ITS-MLP-R, ITS-MAP-F/ITS-MAP-R and ITS-MMD-F/ITS-MMD-R were designed within the internal transcribed spacer (ITS) sequences of ribosomal DNA to target Mlp, Map and Mmd, respectively, and their specificity were confirmed on a wide range of isolates and species. ITS-MLP-F/ITS-MLP-R and ITS-MAP-F/ITS-MAP-R primers proved to be highly specific to Mlp and Map, respectively, whereas ITS-MMD-F/ITS-MMD-R cross-reacted with DNA from M. larici-tremulae and M. pinitorqua. However, these species are not pathogenic on cultivated poplars that all belong to sections Aigeiros and Tacamahaca of the genus Populus. Specific Mmd primers proved to be very sensitive as a positive signal could be obtained with DNA extracts from 6 target urediniospores mixed with 800 000 urediniospores of Mlp. An internal amplification control (IAC) was included to discriminate false negative results due to the potential presence of inhibitory compounds in DNA extracts. ITS-MMD-F/ITS-MMD-R primers are therefore efficient for the detection of the quarantine pathogen Mmd on samples collected on poplar or larch and are fit for use in official tests. This new PCR assay has been used in routine for ten years, and Mmd has hitherto never been detected in commercial poplar nurseries in France