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Abstract Key message Presence of Phytophthora ramorum (Werres, De Cock, and Man in ‘t Veld) in western France was studied after the detection of this invasive pathogen in 2017 in Larix kaempferi (Lamb.) and eradication of the affected stands. P. ramorum was seldom detected in the area of the outbreak in the year following eradication. However, we confirm that P. ramorum can multiply to epidemic level on chestnuts (Castanea sativa Mill.) in the absence of larch (Larix spp.). This represents the major risk in France. Context Phytophthora ramorum is an invasive oomycete that causes significant damage in the USA and Europe. Although the pathogen has been present in nurseries in France since 2002, the first outbreaks in forest stands were identified in 2017 in plantations of Larix kaempferi in two forests in western France (Saint-Cadou and Hanvec). In order to limit the development of the epidemic, neighboring larch stands were clear-cut. Aim This study investigated the presence of P. ramorum in the affected area after the eradication treatment. Methods Larch stands located within a 18-km radius of the reported outbreaks were investigated. We also monitored the native woody hosts present in infected clear-cut larch stands and in the vicinity of seven ornamental nurseries that had been infected by P. ramorum on several occasions in the past. Results Overall, a very limited presence of P. ramorum was detected in 2018–2021. Two new stands of infected L. kaempferi were found close to the main initial outbreak, in Saint-Cadou and Saint-Rivoal. The pathogen was only detected on rhododendrons and chestnut trees (Castanea sativa Mill.) in the vicinity of the outbreaks. In the Saint-Cadou state Forest, an outbreak of the disease developed in 2019–2021 on chestnut trees even though all the mature larch trees had been felled. P. ramorum was also detected near two of the formerly infected ornamental nurseries, on Castanea sativa and on rhododendrons. Conclusion While larches and rhododendrons are uncommon in the forests of north-western France, chestnut trees are present in 21–25% of the forest and therefore represent the major risk for the survival of P. ramorum in the region.

Citrus crops are affected by many fungal diseases. Among them, Citrus Black Spot caused by the ascomycete Phyllosticta citricarpa is particularly economically damaging wherever it occurs. Many other species of Phyllosticta are described on Citrus, but only P. citricarpa is considered a quarantine pest on the European continent. In order to prevent the introduction of this species into Europe, it is essential to have a detection test which can reliably identify it, and not confuse it with other species present on citrus, notably P. paracitricarpa. The latter taxon has recently been described as very close to P. citricarpa, and most detection tests do not allow to distinguish the two species. In this work, we exploited the genomic data of 37 isolates of Phyllosticta spp. from citrus, firstly to assess their phylogenetic relationships, and secondly to search for genomic regions that allowed the definition of species-specific markers of P. citricarpa. Analysis of 51 concatenated genes separated P. citricarpa and P. paracitricarpa in two phylogenetic clades. A locus was selected to define a hydrolysis probe and primers combination that could be used in real-time PCR for the specific detection of the quarantine species, to the exclusion of all others present on Citrus. This test was then thoroughly validated on a set of strains covering a wide geographical diversity, and on numerous biological samples to demonstrate its reliability for regulatory control. The validation data highlighted the need to check the reliability of the test in advance, when a change of reagents was being considered.

[This corrects the article DOI: 10.1371/journal.ppat.1010687.].

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.

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.

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.

Fusarium head blight (FHB) is a major cereal disease caused by a complex of Fusarium species. These species vary in importance depending on climatic conditions, agronomic factors or host genotype. In addition, Fusarium species can release toxic secondary metabolites. These mycotoxins constitute a significant food safety concern as they have health implications in both humans and animals. The Fusarium species involved in FHB differ in their pathogenicity, ability to produce mycotoxins, and fungicide sensitivity. Accurate and exhaustive identification of Fusarium species in planta is therefore of great importance. In this study, using a new set of primers targeting the EF1α gene, the diversity of Fusarium species on cereals was evaluated using Illumina high-throughput sequencing. The PCR amplification parameters and bioinformatic pipeline were optimized with mock and artificially infected grain communities and further tested on 65 field samples. Fusarium species were retrieved from mock communities and good reproducibility between different runs or PCR cycle numbers was be observed. The method enabled the detection of as few as one single Fusarium-infected grain in 10,000. Up to 17 different Fusarium species were detected in field samples of barley, durum and soft wheat harvested in France. This new set of primers enables the assessment of Fusarium diversity by high-throughput sequencing on cereal samples. It provides a more exhaustive picture of the Fusarium community than the currently used techniques based on isolation or species-specific PCR detection. This new experimental approach may be used to show changes in the composition of the Fusarium complex or to detect the emergence of new Fusarium species as far as the EF1α sequence of these species show a sufficient amount of polymorphism in the portion of sequence analyzed. Information on the distribution and prevalence of the different Fusarium species in a given geographical area, and in response to various environmental factors, is of great interest for managing the disease and predicting mycotoxin contamination risks.

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.

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.

Fusarium oxysporum f. sp. cubense (Foc) is one of the most important threats to global banana production. Strategies to control the pathogen are lacking, with plant resistance offering the only long-term solution, if sources of resistance are available. Prevention of introduction of Foc into disease-free areas thus remains a key strategy to continue sustainable banana production. In recent years, strains of Foc affecting Cavendish bananas have destroyed plantations in a number of countries in Asia and in the Middle East, and one African country. One vegetative compatibility group (VCG), 01213/16, is considered the major threat to bananas in tropical and subtropical climatic conditions. However, other genetically related VCGs, such as 0121, may potentially jeopardize banana cultures if they were introduced into disease-free areas. To prevent the introduction of these VCGs into disease-free Cavendish banana-growing countries, a real-time PCR test was developed to accurately detect both VCGs. A previously described putative virulence gene was used to develop a specific combination of hydrolysis probe/primers for the detection of tropical Foc race 4 strains. The real-time PCR parameters were optimized by following a statistical approach relying on orthogonal arrays and the Taguchi method in an attempt to enhance sensitivity and ensure high specificity of the assay. This study also assessed critical performance criteria, such as repeatability, reproducibility, robustness, and specificity, with a large including set of 136 F. oxysporum isolates, including 73 Foc pathogenic strains representing 24 VCGs. The validation data demonstrated that the new assay could be used for regulatory testing applications on banana plant material and can contribute to preventing the introduction and spread of Foc strains affecting Cavendish bananas in the tropics.

Olive trees (cv. Chemlali) showing typical symptoms of wilt and dieback were collected from several areas in Tunisia. Visual diagnostic, isolation and microscopic observations identified the causal pathogen as Verticillium dahliae. The pathotype was determined using specific primers pairs for D (defoliating) and ND (non-defolitating) . Artificial inoculation of olive plants resulted in typical wilting with defoliation, proving that the isolate tested was pathogenic on olive, and this isolate belongs to the D pathotype. This is the first report of the defoliating pathotype causing wilting disease on olive in Tunisia.

Abstract   Chalara fraxinea is a fungus currently threatening ash trees (Fraxinus excelsior) in several European countries. This emerging pathogen was assigned to the EPPO’s alert list and therefore accurate detection and identification tools are needed. Because of its slow growth rate on agar media and the frequent presence of fast-growing saprotrophic fungi within the host tissue, classical isolation techniques are time-consuming and sometimes inefficient. In this study, we used species-specific polymorphisms observed within the internal transcribed spacer region to design a primer pair and a dual-labelled probe to be used in a real-time PCR assay for the detection of C. fraxinea. The test proved to be specific, based on in silico and in vitro assessments, and could detect as little as 20 fg of C. fraxinea DNA. A protocol was developed in order to detect the pathogen directly in plant tissue and proved to be more efficient and rapid than isolation on agar plates. This new tool should be useful both for monitoring and to conduct epidemiology research on this emerging pathogen. [ABSTRACT FROM AUTHOR]

Abstract Since the 1990s, a new Phytophthora species hybrid has been jeopardizing the natural population of alders throughout Europe. This new Phytophthora, P. alni, has been suggested as a natural hybrid between two closely related species of Phytophthora. Little is known about the epidemiology of this pathogen, because its direct isolation is not always satisfactory. In this study we developed three pairs of Polymerase Chain Reaction (PCR) primers derived from Sequence Characterized Amplified Regions (SCAR) that allow discrimination among the three subspecies of P. alni: P. alni subsp. alni, P. alni subsp. uniformis and P. alni subsp. multiformis. These molecular tools were successfully used to detect P. alni directly in different substrates such as infested river water and soil, and necrotic alder bark, without the need for any prior baiting or isolation stages. An Internal Amplification Control (IAC) was included to help discriminate against false negative samples due to the potential presence of inhibitory compounds in DNA extracts. These molecular tools should be useful for epidemiological studies on this emerging disease. [ABSTRACT FROM AUTHOR]

Fusarium Head Blight of small grain cereal is a disease of growing concern in Europe. Along with Microdochium nivale, several species of Fusarium may be associated with the disease, including species that are potentially toxigenic. This paper describes the results of a large scale survey of the variety and frequency of different Fusarium species and M. nivale in France. A total of 749 soft wheat, durum wheat and barley samples were collected and analyzed from 2000 to 2002. The most frequent species isolated were F.graminearum, F. avenaceum and F. poae. The frequency of F. poae seems to have increased while M.nivale and F. culmorum appear less frequent than previously described in France. Other Fusarium species detected in decreasing prevalence were F. tricinctum, F. equiseti, F. acuminatum, F. sambucinum, F.sporotrichioides, F. moniliforme, F. heterosporum, F. subglutinans and F. oxysporum. All the most frequent pathogenic species and also the less pathogenic ones were frequently associated with individual fields. The implications of these associations for the protection of cereals crops and for contamination by mycotoxins are discussed. [ABSTRACT FROM AUTHOR]