Genetic analysis reveals efficient sexual spore dispersal at a fine spatial scale in Armillaria ostoyae, the causal agent of root-rot disease in conifers.

Armillaria ostoyae (sometimes named Armillaria solidipes) is a fungal species causing root diseases in numerous coniferous forests of the northern hemisphere. The importance of sexual spores for the establishment of new disease centres remains unclear, particularly in the large maritime pine plantations of southwestern France. An analysis of the genetic diversity of a local fungal population distributed over 500 ha in this French forest showed genetic recombination between genotypes to be frequent, consistent with regular sexual reproduction within the population. The estimated spatial genetic structure displayed a significant pattern of isolation by distance, consistent with the dispersal of sexual spores mostly at the spatial scale studied. Using these genetic data, we inferred an effective density of reproductive individuals of 0.1-0.3 individuals/ha, and a second moment of parent-progeny dispersal distance of 130-800 m, compatible with the main models of fungal spore dispersal. These results contrast with those obtained for studies of A. ostoyae over larger spatial scales, suggesting that inferences about mean spore dispersal may be best performed at fine spatial scales (i.e. a few kilometres) for most fungal species.

High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries.

Microsatellites (or SSRs: simple sequence repeats) are among the most frequently used DNA markers in many areas of research. The use of microsatellite markers is limited by the difficulties involved in their de novo isolation from species for which no genomic resources are available. We describe here a high-throughput method for isolating microsatellite markers based on coupling multiplex microsatellite enrichment and next-generation sequencing on 454 GS-FLX Titanium platforms. The procedure was calibrated on a model species (Apis mellifera) and validated on 13 other species from various taxonomic groups (animals, plants and fungi), including taxa for which severe difficulties were previously encountered using traditional methods. We obtained from 11,497 to 34,483 sequences depending on the species and the number of detected microsatellite loci ranged from 199 to 5791. We thus demonstrated that this procedure can be readily and successfully applied to a large variety of taxonomic groups, at much lower cost than would have been possible with traditional protocols. This method is expected to speed up the acquisition of high-quality genetic markers for nonmodel organisms.

Temporal and Spatial Dynamics of Primary and Secondary Infection by Armillaria ostoyae in a Pinus pinaster Plantation.

ABSTRACT Epidemiological investigations were performed in a 3-ha maritime pine (Pinus pinaster) plantation established on a site heavily infested by Armillaria ostoyae. Geostatistics were used to examine the density and the distribution of the initial inoculum. Disease dynamics were monitored for 17 years after planting. On the whole site, the cumulative mortality rate reached 35% over this period, plateauing at 12 years. Disease progress curves differed according to the density of the initial inoculum, although in all the cases, the Gompertz model described the epidemics well. The epidemiological contributions of both primary (initially colonized stumps) and secondary inoculum (newly dead pines) were evaluated by analyzing their spatial relation to annual mortality. Newly dead pines acted as secondary inoculum from year 3 and their role increased with time. When the initial inoculum density was low, the contribution of secondary inoculum to epidemic development increased faster and halted sooner than when the density of primary inoculum was high. Regardless of its density, the primary inoculum acted throughout the dynamic phase of the epidemic. When the inoculum density was low, the probability of mortality during the first 6 years of the epidemic depended on the tree distance from the nearest stumps colonized by Armillaria sp. When the inoculum density was high, the probability of mortality was higher and not related to the distance between trees and colonized stumps.