A point mutation and large deletion at the candidate avirulence locus AvrMlp7 in the poplar rust fungus correlate with poplar RMlp7 resistance breakdown.

The deployment of plant varieties carrying resistance genes (R) exerts strong selection pressure on pathogen populations. Rapidly evolving avirulence genes (Avr) allow pathogens to escape R-mediated plant immunity through a variety of mechanisms, leading to virulence. The poplar rust fungus Melampsora larici-populina is a damaging pathogen of poplars in Europe. It underwent a major adaptive event in 1994, with the breakdown of the poplar RMlp7 resistance gene. Population genomics studies identified a locus in the genome of M. larici-populina that probably corresponds to the candidate avirulence gene AvrMlp7. Here, to further characterize this effector, we used a population genetics approach on a comprehensive set of 281 individuals recovered throughout a 28-year period encompassing the resistance breakdown event. Using two dedicated molecular tools, genotyping at the candidate locus highlighted two different alterations of a predominant allele found mainly before the resistance breakdown: a nonsynonymous mutation and a complete deletion of this locus. This results in six diploid genotypes: three genotypes related to the avirulent phenotype and three related to the virulent phenotype. The temporal survey of the candidate locus revealed that both alterations were found in association during the resistance breakdown event. They pre-existed before the breakdown in a heterozygous state with the predominant allele cited above. Altogether, these results suggest that the association of both alterations at the candidate locus AvrMlp7 drove the poplar rust adaptation to RMlp7-mediated immunity. This study demonstrates for the first time a case of adaptation from standing genetic variation in rust fungi during a qualitative resistance breakdown.

Evolution of morphological but not aggressiveness-related traits following a major resistance breakdown in the poplar rust fungus, Melampsora larici-populina.

Crop varieties carrying qualitative resistance to targeted pathogens lead to strong selection pressure on parasites, often resulting in resistance breakdown. It is well known that qualitative resistance breakdowns modify pathogen population structure but few studies have analyzed the consequences on their quantitative aggressiveness-related traits. The aim of this study was to characterize the evolution of these traits following a resistance breakdown in the poplar rust fungus, Melampsora larici-populina. We based our experiment on three temporal populations sampled just before the breakdown event, immediately after and four years later. First, we quantified phenotypic differences among populations for a set of aggressiveness traits on a universally susceptible cultivar (infection efficiency, latent period, lesion size, mycelium quantity, and sporulation rate) and one morphological trait (mean spore volume). Then, we estimated heritability to establish which traits could be subjected to adaptive evolution and tested for evidence of selection. Our results revealed significant changes in the morphological trait but no variation in aggressiveness traits. By contrast, recent works have demonstrated that quantitative resistance (initially assumed more durable) could be eroded and lead to increased aggressiveness. Hence, this study is one example suggesting that the use of qualitative resistance may be revealed to be less detrimental to long-term sustainable crop production.

Defense Compounds Rather Than Nutrient Availability Shape Aggressiveness Trait Variation Along a Leaf Maturity Gradient in a Biotrophic Plant Pathogen.

Foliar pathogens face heterogeneous environments depending on the maturity of leaves they interact with. In particular, nutrient availability as well as defense levels may vary significantly, with opposing effects on the success of infection. The present study tested which of these factors have a dominant effect on the pathogen's development. Poplar leaf disks of eight maturity levels were inoculated with the poplar rust fungus Melampsora larici-populina using an innovative single-spore inoculation procedure. A set of quantitative fungal traits (infection efficiency, latent period, uredinia size, mycelium quantity, sporulation rate, sporulation capacity, and spore volume) was measured on each infected leaf disk. Uninfected parts of the leaves were analyzed for their nutrient (sugars, total C and N) and defense compounds (phenolics) content. We found that M. larici-populina is more aggressive on more mature leaves as indicated by wider uredinia and a higher sporulation rate. Other traits varied independently from each other without a consistent pattern. None of the pathogen traits correlated with leaf sugar, total C, or total N content. In contrast, phenolic contents (flavonols, hydroxycinnamic acid esters, and salicinoids) were negatively correlated with uredinia size and sporulation rate. The pathogen's fitness appeared to be more constrained by the constitutive plant defense level than limited by nutrient availability, as evident in the decrease in sporulation.

The escalatory Red Queen: Population extinction and replacement following arms race dynamics in poplar rust.

Host-parasite systems provide convincing examples of Red Queen co-evolutionary dynamics. Yet, a key process underscored in Van Valen's theory - that arms race dynamics can result in extinction - has never been documented. One reason for this may be that most sampling designs lack the breadth needed to illuminate the rapid pace of adaptation by pathogen populations. In this study, we used a 25-year temporal sampling to decipher the demographic history of a plant pathogen: the poplar rust fungus, Melampsora larici-populina. A major adaptive event occurred in 1994 with the breakdown of R7 resistance carried by several poplar cultivars widely planted in Western Europe since 1982. The corresponding virulence rapidly spread in M. larici-populina populations and nearly reached fixation in northern France, even on susceptible hosts. Using both temporal records of virulence profiles and temporal population genetic data, our analyses revealed that (i) R7 resistance breakdown resulted in the emergence of a unique and homogeneous genetic group, the so-called cultivated population, which predominated in northern France for about 20 years, (ii) selection for Vir7 individuals brought with it multiple other virulence types via hitchhiking, resulting in an overall increase in the population-wide number of virulence types and (iii) - above all - the emergence of the cultivated population superseded the initial population which predominated at the same place before R7 resistance breakdown. Our temporal analysis illustrates how antagonistic co-evolution can lead to population extinction and replacement, hence providing direct evidence for the escalation process which is at the core of Red Queen dynamics.

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.

Digital photography as an operational tool for assessing corticosteroid-induced lipodystrophy.

BACKGROUND: Corticosteroid-induced lipodystrophy (CIL) is exclusively diagnosed in a subjective manner. OBJECTIVE: To evaluate the reliability of digital photographs in the diagnosis of CIL. METHODS: All consecutive patients starting long-term, high dosage corticosteroid therapy were photographed at baseline and after 3 months of therapy. At the end of the study, 3 physicians with expertise in corticosteroids classified patients as lipodystrophic yes/no/unclassifiable. Photographs analyses performed by 9 medical readers and evaluation of CIL using visual analog scale (VAS) performed during the M3 visit were compared to this classification. RESULTS: Eighty-eight patients were monitored. Fifty of them were classified by the 3 experts as lipodystrophic and 30 as not lipodystrophic (8 were unclassifiable). Their intra- and inter-observer agreements were moderate or fair (kappa coefficientor=0.75) when M3 photographs were analysed beside baseline ones. By comparison with expert consensus, only 3 out of 4 patients were correctly classified using VAS. The AUROC curve and inter-observer agreement significantly improved with experience for the 9 non-experts. CONCLUSION: The use of digital photographs do better than VAS to evaluate CIL. The accuracy of diagnosis improves with experience. Morphological changes are more important than morphological phenotype.

Occupational exposure to solvents and gender-related risk of systemic sclerosis: a metaanalysis of case-control studies.

OBJECTIVE: In 2001 a metaanalysis reported an excess risk of systemic sclerosis (SSc) related to solvents exposure. The magnitude of risk varied among studies and sources of heterogeneity have not been investigated due to a lack of statistical power. We conducted a new metaanalysis to identify features associated with the magnitude of SSc risk in patients exposed to solvents. METHODS: We searched 4 databases (Medline, Pascal, Pascal Biomed, Francis). Inclusion criteria were: case-control study, occupational exposure to solvents (OES) assessed by questionnaire and summarized to "any solvent" or "any organic solvent," SSc defined by the American College of Rheumatology or the consultant's criteria. The quality of studies within this metaanalysis was scored according to the Newcastle-Ottawa scale. Odds ratios (OR) were adjusted for the "publication bias" and validated by a sensitivity analysis. Subgroup analyses investigated the effect of gender, quality of studies, and the type of controls. RESULTS: Among 11 studies (1291 patients and 3435 controls), 9 involved a majority of women (76.2 to 100%), while 2 involved men only. The risk of SSc associated with OES was variable among studies (p for heterogeneity = 0.01) and overrepresentation of higher OR values in smaller studies (p = 0.003) suggested "publication bias." SSc was associated with OES (OR 2.4; 95% CI 1.7-3.4; p < 0.0001), including after adjusting for bias (OR 1.8; 95% CI 1.2-2.5; p = 0.002). The relative risk was higher (p = 0.03) in men (OR 3.0; 95% CI 1.9-4.6; p < 0.0001) than in women (OR 1.8; 95% CI 1.5-2.1; p < 0.0001). CONCLUSION: Whereas SSc affects women predominantly, among subjects with occupational exposure to solvents, men are at higher risk than women for the disease.