Claude Husson

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.

Partial resistance to Melampsora larici-populina leaf rust in hybrid poplars: Genetic variability in inoculated excised leaf disk bioassay and relationship with complete resistance

ABSTRACT Inoculated excised leaf disk technique allows decomposition of poplar partial resistance to Melampsora larici-populina leaf rust into key epidemiological components such as latent period (LP), uredinia number (UN), uredinia size (US), and spore production (SP) for a given M. larici-populina strain under controlled environmental conditions. Three hundred thirty-six genotypes from an interspecific Populus deltoides x P. trichocarpa F(1) progeny segregating for complete resistance to M. larici-populina strain 93ID6 were inoculated with M. larici-populina strain 93CV1. This strain was able to infect the whole family, except few probable recombinants. LP, final UN, and final US after one infectious cycle proved to be relevant complementary descriptors of partial resistance. Area under the disease progress curve and other parameters of uredinia appearance dynamics did not yield additional information. Indirect assessment of SP by US scoring was reliable and easy to access compared with direct spore counting. UN was the only trait for which a doubling of the inoculum pressure level had a significant effect, leading to greater differentiation between genotypes. Consistent with previous studies is the clear relationship between presence of complete resistance against M. larici-populina strain 93ID6 and higher partial resistance to M. larici-populina strain 93CV1 (32% longer LP, 76% smaller UN, and 34% smaller US). In the subpopulation compatible with 93ID6, bimodal distribution of genotypic means for US suggested implication of a major gene inherited from the P. trichocarpa parent. Residual variation was noted for the three epidemiological components, suggesting that additional genes might condition these quantitative traits.

Comparison of genetic and virulence diversity of Melampsora larici-populina populations on wild and cultivated poplar and influence of the alternate host

ABSTRACT The aims of this study were, first, to compare the genetic and virulence diversity between populations of the rust fungus Melampsora larici-populina on wild and cultivated poplar stands and, second, to investigate the influence of the presence of the alternate host of the pathogen, larch, on which its sexual reproduction occurs, on these diversities. Nine French M. larici-populina populations collected from poplar trees in autumn and four populations collected from larch trees during the following spring were analyzed using both virulence factors and neutral markers. In all, 30 pathotypes were identified within the 13 populations studied. The pathotypic structure clearly distinguished the cultivated stands with high richness and complexity from the wild stands with low richness and complexity. High linkage disequilibria between virulences indicated preferential virulence associations, probably due to selection by the host. In all, 19 random amplified polymorphic DNA (RAPD) markers were used, which revealed a very high genetic diversity in the 743 isolates analyzed. The nine populations from poplar appeared moderately differentiated, indicating long-distance gene flow, and no isolation by distance was found. Linkage disequilibria between RAPD markers generally were low, indicating frequent recombination, but they were not lower in populations located near larch, probably due to long-distance dispersal.

Strong genetic differentiation between North American and European populations of Phytophthora alni subsp. uniformis.

Alder decline caused by Phytophthora alni has been one of the most important diseases of natural ecosystems in Europe during the last 20 years. The emergence of P. alni subsp. alni -the pathogen responsible for the epidemic-is linked to an interspecific hybridization event between two parental species: P. alni subsp. multiformis and P. alni subsp. uniformis. One of the parental species, P. alni subsp. uniformis, has been isolated in several European countries and, recently, in North America. The objective of this work was to assess the level of genetic diversity, the population genetic structure, and the putative reproduction mode and mating system of P. alni subsp. uniformis. Five new polymorphic microsatellite markers were used to contrast both geographical populations. The study comprised 71 isolates of P. alni subsp. uniformis collected from eight European countries and 10 locations in North America. Our results revealed strong differences between continental populations (Fst = 0.88; Rst = 0.74), with no evidence for gene flow. European isolates showed extremely low genetic diversity compared with the North American collection. Selfing appears to be the predominant mating system in both continental collections. The results suggest that the European P. alni subsp. uniformis population is most likely alien and derives from the introduction of a few individuals, whereas the North American population probably is an indigenous population.

An evolutionary ecology perspective to address forest pathology challenges of today and tomorrow

Key messageIncreasing human impacts on forests, including unintentional movement of pathogens, climate change, and large-scale intensive plantations, are associated with an unprecedented rate of new diseases. An evolutionary ecology perspective can help address these challenges and provide direction for sustainable forest management.ContextForest pathology has historically relied on an ecological approach to understand and address the practical management of forest diseases. A widening of this perspective to include evolutionary considerations has been increasingly developed in response to the rising rates of genetic change in both pathogen populations and tree populations due to human activities.AimsHere, five topics for which the evolutionary perspective is especially relevant are highlighted.ResultsThe first relates to the evolutionary diversity of fungi and fungal-like organisms, with issues linked to the identification of species and their ecological niches. The second theme deals with the evolutionary processes that allow forest pathogens to adapt to new hosts after introductions or to become more virulent in homogeneous plantations. The third theme presents issues linked to disease resistance in tree breeding programs (e.g., growth-defense trade-offs) and proposes new criteria and methods for more durable resistance. The last two themes are dedicated to the biotic environment of the tree–pathogen system, namely, hyperparasites and tree microbiota, as possible solutions for health management.ConclusionWe conclude by highlighting three major conceptual advances brought by evolutionary biology, i.e., that (i) “not everything is everywhere”, (ii) evolution of pathogen populations can occur on short time scales, and (iii) the tree is a multitrophic community. We further translate these into a framework for immediate policy recommendations and future directions for research.

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.

Risk Factors for the Phytophthora-Induced Decline of Alder in Northeastern France

ABSTRACT A lethal disease of common alder caused by Phytophthora alni, a new hybrid pathogen, has been spreading in Europe since the early 1990s. In 2004, we conducted an epidemiological survey in northeastern France to determine disease frequency and to investigate the impact of environmental factors on disease prevalence. Seventy-eight plots in the Rhin-Meuse basin were investigated. The survey was structured to enable critical examination of the possible impact of nitrogen pollution of the river water on disease prevalence. P. alni-induced alder decline was common throughout northeastern France. Altogether, disease was found in 80% of the plots containing alder, with 16% of all the alders affected. Striking differences existed between watercourse types. Lower proportions of diseased alders were found in watercourse types with rapid water flow, such as mountain streams of the Vosges and piedmont or watercourses on steep calcareous slopes, than in the slow watercourses of the low-lying valleys of the calcareous plateaus and of the clayey plains. Disease prevalence was not related to the total oxidized nitrogen concentration of the water. However, prevalence increased with the mean summer temperature of the river water and where clayey soils were found in the river banks. The results of this work can be used for the assessment of P. alni-induced alder decline risks in affected European countries and in areas where the disease could be introduced.

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.

A statistical model to detect asymptomatic infectious individuals with an application in the Phytophthora alni-induced alder decline.

In some diseases-in particular, tree root infection-stages of infection and inoculum production level and timing are not readily observable because of uncertainty or time lags in symptom appearance. Here, we pose a criterion, based on relative hazard of disease symptoms, to discriminate between healthy and asymptomatic infected individuals. We design a statistical procedure to estimate the criterion for a 6-year survey of alder decline along a northeastern French river. Individual tree symptom hazard was modeled with Coxs regression model, taking estimation of local infection pressure as a risk factor. From an inoculum production experiment, we thereafter assessed the inoculum production level of target trees, including symptomatic and asymptomatic trees ranked according to their symptoms hazard. Using receiver operating characteristic methods, we first evaluated the criterion performance and determined the discrimination threshold to sort out asymptomatic individuals into healthy and infected. Then, we highlighted the fact that the infected asymptomatic trees were among the major inoculum producers whereas severely declining and dead trees were found to be poor inoculum sources.

First report of Phytophthora ramorum on ornamental plants in France.

In April 2002, Phytophthora ramorum was associated with twig blight and brown spots on Rhododendron spp. leaves from a nursery in France. The isolate was identified by its morphological characters on V8 agar: slow growth, deciduous and semipapillate sporangia, and abundant production of large chlamydospores (3). The identification was confirmed by ITS rDNA sequencing. During 2002, P. ramorum was also isolated from diseased Viburnum tinus and V. × bodnantense plants exhibiting symptoms of wilting and stem base discoloration. Subsequently, repeated surveys for P. ramorum were carried out in nurseries and areas surrounding nurseries throughout France. Since 2004, a large range of known hosts were investigated in approximately 2,000 nurseries and 200 other sites each year. P. ramorum was detected exclusively in nurseries at 29 locations in 2002, 9 in 2003, 23 in 2004, 17 in 2005, and 19 in 2006. Rhododendron spp. and occasionally V. tinus were the major hosts. In addition, the pathogen was detected for the first time on Pieris japonica in two nurseries in 2005 and on Camellia sp. in one nursery in 2006 from plants exhibiting leaf and twig blight. In both cases, P. ramorum had already been detected on Rhododendron spp. in the same nurseries. Most of the infected plants were found in northwestern France (Bretagne and Pays-de-la-Loire), or came from this region, which is the main rhododendron-growing area in France. In some cases, plants were imported from Belgium or the Netherlands. P. ramorum was also detected in a nursery in soil close to diseased Rhododendron spp. plants and pond water used for irrigation by using a combination of baiting with Rhododendron spp. leaves and PCR assay with species-specific primers (1). Overall, approximately 1% of the investigated nurseries were found positive each year, and this ratio was quite stable from 2004 to 2006. To date, P. ramorum has not been detected outside of nurseries, although many surveys were conducted on the west coast of France where the risk is considered to be high because of a favorable mild and humid climate and the presence of suitable hosts. In addition, 78 isolates of P. ramorum collected between 2002 and 2004 on Rhododendron spp. and V. tinus were found to be of A1 mating type based on pairings with P. cryptogea A1 and A2 mating types (2). References: (1) K. J. Hayden et al. Phytopathology 94:1075, 2004. (2) S. Werres and B. Zielke J. Plant Dis. Prot. 110:129, 2003. (3) S. Werres et al. Mycol. Res. 105:1155, 2001.