Renaud Ioos

Chalara fraxinea is an invasive pathogen in France

Decline induced by Chalara fraxinea is an emerging disease that severely affects ash stands in Europe. The disease appears to have an invasive spread from East to West of Europe in the last decade. The teleomorphic stage, Hymenoscyphus pseudoalbidus, that occurs as apothecia on ash rachis in the litter was recently described. The origin of ash decline remains unclear as a cryptic species, H. albidus, a long-established fungus in Europe, could be present in the same niche, and as in Switzerland, H. pseudoalbidus was shown to have been present long before the recent epidemic outbreak. In France, the emerging disease is very recent and clearly restrained to Northeastern France. We thus collected isolates from infected hosts and from apothecia/ash rachis both inside and outside the infected area in France in order to compare them on the basis of pathogenicity towards ash seedlings and sequences of the ITS regions and of three single-copy genes. We showed that two population types exhibiting about 2% base pair polymorphism in the sequences analysed were present in Northern France. The first type, corresponding to H. pseudoalbidus, was present on rachis and infected hosts only in Northeastern France and showed strong pathogenicity towards ash seedlings in inoculation tests. By contrast, the second type, which corresponds to H. albidus, was present throughout Northern France and showed no pathogenicity towards ash seedlings. Our study confirms the results of Queloz et al. (2010) who presented molecular evidences for the existence of two cryptic species, H. albidus and H. pseudoalbidus. The results strongly suggest that Chalara fraxinea/H. pseudoalbidus is a recent invader in France.

Genomic Variation within Monilinia laxa, M. fructigena and M. fructicola, and Application to Species Identification by PCR

Brown rot and twig canker of fruit trees are caused by Monilinia laxa, M. fructigena and M. fructicola. The Internal Transcribed Spacer (ITS) between the 18S and the 28S rRNA genes of four M. laxa and four M. fructigena isolates collected in France was amplified by Polymerase Chain Reaction (PCR) using universal primers and sequenced. Multiple alignment of the ITS sequences and comparison with published sequences revealed very little intraspecific variation and a low interspecific polymorphism clustered in two regions. Species-specific PCR primers were designed to amplify a 356 bp fragment for each of the three species. The specificity of the three primer pairs was successfully tested with a collection of 17 M. laxa, 18 M. fructigena and 6 M. fructicola isolates collected from different hosts and different countries, unequivocally confirming the identification of each isolate based on morphological and cultural traits. Using stringent PCR conditions, no cross-reaction was observed with any of the isolates tested. The specificity of the PCR assays was also successfully confirmed with DNA extracted from different fungal species, either phylogenetically close to the genus Monilinia or commonly found on diseased fruits. Using this new reliable technique, doubtful isolates can be directly identified in a single PCR run. Moreover, detection and identification of the Monilinia species were successfully achieved directly on diseased fruits. This simple and rapid method can be particularly useful to detect M. fructicola which is a listed quarantine fungus in all European countries.

Development, Comparison, and Validation of Real-Time and Conventional PCR Tools for the Detection of the Fungal Pathogens Causing Brown Spot and Red Band Needle Blights of Pine

Dothistroma pini, D. septosporum, and Lecanosticta acicola are fungal pathogens that cause severe foliage diseases in conifers. All three pathogens are listed as quarantine organisms in numerous countries throughout the world and, thus, are subject to specific monitoring. Detection and identification of these pathogens still often relies on cumbersome and unsatisfactory methods that are based upon the morphological characterization of fungal fruiting bodies and conidia. In this study, we present the development of several new molecular tools that enable a rapid and specific in planta detection of each of these pathogens. Several DNA extraction procedures starting from infected needles were compared and four commercial DNA extraction kits provided DNA of satisfactory quality for amplification by polymerase chain reaction (PCR). In addition, we developed several sets of conventional PCR primers, dual-labeled probes (DLPs), and duplex-scorpion probes (DSPs), all of which targeted each pathogen. Their ability to detect the pathogens in a series of naturally infected needle samples was compared. The quadruplex DLP real-time assay proved to be more sensitive than the DSP assay and conventional PCR but the two real-time probe formats yielded identical results in the naturally infected samples. Both real-time assays proved to be significantly superior to the technique of humid chamber incubation, which often failed to produce spores for the accurate identification of the pathogens.

Evidence for homoploid speciation in Phytophthora alni supports taxonomic reclassification in this species complex.

Alder decline has been a problem along European watercourses since the early 1990s. Hybridization was identified as the main cause of this emerging disease. Indeed, the causal agent, a soil-borne pathogen named Phytophthora alni subsp. alni (Paa) is the result of interspecific hybridization between two taxa, Phytophthora alni subsp. multiformis (Pam) and Phytophthora alni subsp. uniformis (Pau), initially identified as subspecies of Paa. The aim of this work was to characterize the ploidy level within the P. alni complex that is presently poorly understood. For that, we used two complementary approaches for a set of 31 isolates of Paa, Pam and Pau: (i) quantification of allele copy number of three single-copy nuclear genes using allele-specific real-time PCR and (ii) comparison of the genome size estimated by flow cytometry. Relative quantification of alleles of the three single-copy genes showed that the copy number of a given allele in Paa was systematically half that of its parents Pau or Pam. Moreover, DNA content estimated by flow cytometry in Paa was equal to half the sum of those in Pam and Pau. Our results therefore suggest that the hybrid Paa is an allotriploid species, containing half of the genome of each of its parents Pam and Pau, which in turn are considered to be allotetraploid and diploid, respectively. Paa thus results from a homoploid speciation process. Based on published data and on results from this study, a new formal taxonomic name is proposed for the three taxa Paa, Pam and Pau which are raised to species status and renamed P. ×alni, P. ×multiformis and P. uniformis, respectively.

Is the Emergence of Dothistroma Needle Blight of Pine in France Caused by the Cryptic Species Dothistroma pini

Dothistroma needle blight (DNB) emerged in France in the past 15 years. This disease is induced by two closely related species: Dothistroma septosporum and D. pini. Although both species are currently present in France, only D. septosporum was reported in the past. We investigated whether a recent arrival of D. pini in France could be a cause of the DNB emergence. We analyzed herbarium specimens of pine needles with DNB symptoms using polymerase chain reaction techniques to study the past frequency of D. pini in France. We also determined the present distribution within the country of D. septosporum and D. pini and compared it with the spatial pattern of DNB reported in the Département de la Santé des Forêts (DSF; French forest health monitoring agency) database. Although D. pini was detected on herbarium specimens from 1907 and 1965, it was not frequent in France in the past. Today, it is frequent, although not present throughout the country, being absent from the north and the east. There is no relationship between the D. pini distribution in France and the spatial pattern of DNB reported in the DSF database. Thus, the emergence of DNB in France cannot be explained by a recent arrival of D. pini.

An Optimized Duplex Real-Time PCR Tool for Sensitive Detection of the Quarantine Oomycete Plasmopara halstedii in Sunflower Seeds

Plasmopara halstedii, the causal agent of downy mildew of sunflower, is an oomycete listed as a quarantine pathogen. This obligate parasite resides in a quiescent state in seeds of sunflower and can be spread from seed production areas to areas of crop production by international seed trade. To prevent the spread or the introduction of potentially new genotypes or fungicide-tolerant strains, an efficient method to detect P. halstedii in sunflower seed is required. This work reports the optimization of a real-time detection tool that targets the pathogen within sunflower seeds, and provides statistically validated data for that tool. The tool proved to be specific and inclusive, based on computer simulation and in vitro assessments, and could detect as few as 45 copies of target DNA. A fully optimized DNA extraction protocol was also developed starting from a sample of 1,000 sunflower seeds, and enabled the detection of <1 infected seed/1,000 seeds. To ensure reliability of the results, a set of controls was used systematically during the assays, including a plant-specific probe used in a duplex quantitative polymerase chain reaction that enabled the assessment of the quality of each DNA extract.

Usefulness of single copy genes containing introns in Phytophthora for the development of detection tools for the regulated species P. ramorum and P. fragariae

Introns are generally highly polymorphic regions within genes and were proven to be of great interest for discriminating among phylogenetically-close Phytophthora species. Phytophthora ramorum and P. fragariae are considered as quarantine pathogens by the European Union and accurate detection tools are therefore necessary for their monitoring. From introns located in different single copy genes (GPA1, RAS-like, and TRP1), we developed a series of PCR primers specific to P. ramorum and P. fragariae. The specificity of these primers was successfully checked with a wide collection of Phytophthora isolates and a protocol was developed to detect both pathogens directly in infected plant tissues. These genes should be of particular interest for the development of additional species-specific detection tools within the Phytophthora genus.

Detection of plant pathogens using real‐time PCR: how reliable are late Ct values?

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.

Distribution and Expression of Elicitin Genes in the Interspecific Hybrid Oomycete Phytophthora alni

ABSTRACT Phytophthora alni subsp. alni, P. alni subsp. multiformis, and P. alni subsp. uniformis are responsible for alder disease in Europe. Class I and II elicitin gene patterns of P. alni subsp. alni, P. alni subsp. multiformis, P. alni subsp. uniformis, and the phylogenetically close species P. cambivora and P. fragariae were studied through mRNA sequencing and 3′ untranslated region (3′UTR)-specific PCRs and sequencing. The occurrence of multiple 3′UTR sequences in association with identical elicitin-encoding sequences in P. alni subsp. alni indicated duplication/recombination events. The mRNA pattern displayed by P. alni subsp. alni demonstrated that elicitin genes from all the parental genomes are actually expressed in this allopolyploid taxon. The complementary elicitin patterns resolved confirmed the possible involvement of P. alni subsp. multiformis and P. alni subsp. uniformis in the genesis of the hybrid species P. alni subsp. alni. The occurrence of multiple and common elicitin gene sequences throughout P. cambivora, P. fragariae, and P. alni sensu lato, not observed in other Phytophthora species, suggests that duplication of these genes occurred before the radiation of these species.

First Report of Blight Disease on Buxus Caused by Cylindrocladium buxicola in France

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.