Benoit Marçais

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.

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.

Leaf and root-associated fungal assemblages do not follow similar elevational diversity patterns.

The diversity of fungi along environmental gradients has been little explored in contrast to plants and animals. Consequently, environmental factors influencing the composition of fungal assemblages are poorly understood. The aim of this study was to determine whether the diversity and composition of leaf and root-associated fungal assemblages vary with elevation and to investigate potential explanatory variables. High-throughput sequencing of the Internal Transcribed Spacer 1 region was used to explore fungal assemblages along three elevation gradients, located in French mountainous regions. Beech forest was selected as a study system to minimise the host effect. The variation in species richness and specific composition was investigated for ascomycetes and basidiomycetes assemblages with a particular focus on root-associated ectomycorrhizal fungi. The richness of fungal communities associated with leaves or roots did not significantly relate to any of the tested environmental drivers, i.e. elevation, mean temperature, precipitation or edaphic variables such as soil pH or the ratio carbon∶nitrogen. Nevertheless, the ascomycete species richness peaked at mid-temperature, illustrating a mid-domain effect model. We found that leaf and root-associated fungal assemblages did not follow similar patterns of composition with elevation. While the composition of the leaf-associated fungal assemblage correlated primarily with the mean annual temperature, the composition of root-associated fungal assemblage was explained equally by soil pH and by temperature. The ectomycorrhizal composition was also related to these variables. Our results therefore suggest that above and below-ground fungal assemblages are not controlled by the same main environmental variables. This may be due to the larger amplitude of climatic variables in the tree foliage compared to the soil environment.

Effect of poplar genotypes on mycorrhizal infection and secreted enzyme activities in mycorrhizal and non-mycorrhizal roots

The impact of ectomycorrhiza formation on the secretion of exoenzymes by the host plant and the symbiont is unknown. Thirty-eight F1 individuals from an interspecific Populus deltoides (Bartr.)×Populus trichocarpa (Torr. & A. Gray) controlled cross were inoculated with the ectomycorrhizal fungus Laccaria bicolor. The colonization of poplar roots by L. bicolor dramatically modified their ability to secrete enzymes involved in organic matter breakdown or organic phosphorus mobilization, such as N-acetylglucosaminidase, β-glucuronidase, cellobiohydrolase, β-glucosidase, β-xylosidase, laccase, and acid phosphatase. The expression of genes coding for laccase, N-acetylglucosaminidase, and acid phosphatase was studied in mycorrhizal and non-mycorrhizal root tips. Depending on the genes, their expression was regulated upon symbiosis development. Moreover, it appears that poplar laccases or phosphatases contribute poorly to ectomycorrhiza metabolic activity. Enzymes secreted by poplar roots were added to or substituted by enzymes secreted by L. bicolor. The enzymatic activities expressed in mycorrhizal roots differed significantly between the two parents, while it did not differ in non-mycorrhizal roots. Significant differences were found between poplar genotypes for all enzymatic activities measured on ectomycorrhizas except for laccases activity. In contrast, no significant differences were found between poplar genotypes for enzymatic activities of non-mycorrhizal root tips except for acid phosphatase activity. The level of enzymes secreted by the ectomycorrhizal root tips is under the genetic control of the host. Moreover, poplar heterosis was expressed through the enzymatic activities of the fungal partner.

European oak powdery mildew: impact on trees, effects of environmental factors, and potential effects of climate change

Abstract• ContextPowdery mildew is one of the most common diseases of oaks in Europe. After alarming reports in the beginning of the twentieth century following the presumed introduction of the invasive fungus, the disease has become familiar to foresters. However, its impact may vary greatly according to intrinsic and extrinsic factors.• AimsWe aimed at providing updated and synthesised information on the impact of powdery mildew on oak and on the effects of environment on disease.• MethodsA comprehensive literature review was performed, including old reports of the early epidemics to more recent data.• ResultsTree growth patterns are of critical importance to explain the severity of the disease and the differences between juvenile and mature trees. A critical element, especially for infection of mature trees, is the availability of spores during the production of the first leaf flush. High disease impact is often related to modified growth patterns, either by environmental factors (insects or frost) or silvicultural practices (e.g., coppicing).• ConclusionPowdery mildew can have important impacts in natural oak regenerations and a significant role in decline of mature trees. Climate change might influence the disease severity mainly by altering the host pathogen phenological synchrony. Process-based models are required for reliable predictions.

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.

Prediction and mapping of the impact of winter temperature on the development of Phytophthora cinnamomi-induced cankers on red and pedunculate oak in France

ABSTRACT Phytophthora cinnamomi is the causal agent of a perennial canker that develops on the lower bole on northern red oak and pedunculate oak. The disease has a limited range in Europe, being reported only in southwest France. This limited distribution is probably linked to the susceptibility of P. cinnamomi to frost. A model was developed in previous work to estimate the impact of temperatures of <0 degrees C on the winter survival of P. cinnamomi in trunk cortical tissues and on the subsequent development of cankers. In this article, we report the use of this model to simulate canker development in 503 locations across France during a 30-year period. The predicted canker extension decreased sharply when the median P. cinnamomi winter survival index decreased from 0.95 to 0.65, with cankers that poorly developed when the median survival index was lower than 0.5 to 0.6. The actual incidence of the disease in 192 stands located across southwest France was compared with that of the model outputs. Both presence of disease in stands and frequency of cankered trees in infected stands, but not canker size on infected trees, were strongly related to the median P. cinnamomi survival index. No disease was present in stands with median survival index lower than 0.65, and the frequency of cankered trees in infected stands remained very low in stands with a median survival index between 0.65 and 0.70. Aspect was an additional factor explaining disease incidence, while the effect of elevation was likely due to its effect on winter temperatures. Maps of winter suitability to P. cinnamomi-induced cankers on oaks in France are presented.