Pathogens introduction as a collateral effect of military activity.

The introduction of exotic tree diseases has occurred many times over the past 150 years. The most common pathways for introduction of pathogens are movement of infected planting stock2,3 or infested wood4. These pathways are well understood and monitored by regulatory agencies. Less understood pathways, however, exist for the introduction of exotic pathogens. Heterobasidion species are important root pathogens with circumboreal distribution. H. annosum was found to be consistently associated with high mortality of stone pine (Pinus pinea) in the forest of the Presidential Estate of Castelporziano, near Rome (Italy). Several pure cultures of the pathogen were obtained from fertile fruiting bodies, DNAs were extracted, and PCR amplification using H. annosum-specific primers showed the presence of a mitochondrial insertion reported from North America, but known to be absent in Europe5. This unusual finding prompted us to sequence portions of the insertion and of three additional loci from seven Castelporziano and 97 Heterobasidion individuals of worldwide distribution. Maximum parsimony analysis of the nuclear glyceraldehydes 3-phosphate dehydrogenase was performed using PAUP, with gaps and insertions counted as single characters. This analysis differentiated North American from European populations with a bootstrap value of 100, and showed that the Castelporziano individuals always clustered within H. annosum populations infecting North American pines. Additional analyses of the nuclear elongation factor 1-alpha, the mitochondrial ATPase subunit 6, and of the insertion in the mitochondrial ribosomal operon, never before reported from Europe, all clustered the Castelporziano individuals with H. annosum populations from eastern North America. Inferences for this study were made from analyses of a total of 2236 base pairs, including 127 parsimony informative characters, from four unlinked loci. Bootstrap values were calculated using the Fast Step algorithm in PAUP for 1000 replicates. Trees were constructed using three closely related taxa as outgroups. The data support the hypothesis that the Heterobasidion population at Castelporziano originated from eastern North America. Native Heterobasidion populations rely on sexual reproduction and are comprised of several different genotypes7,8. Individuals from Castelporziano were all different genotypes based on microsatellite fingerprinting and on somatic self-compatibility tests. These findings suggest that this introduced population has become successfully established in the Castelporziano woodlands. The exotic disease is not widespread outside the Presidential Estate, and its further spread could be limited by a disease mitigation/eradication strategy. The Estate is not a park but a hunting preserve covered by woodlands, it has been closed to the public for centuries, and is comprised of an exclusively native Italian flora with the exception of a few Eucalyptus trees9. The question remains: how was Heterobasidion introduced from North America? The 5th U.S. Army regiments occupied the Estate grounds for several months during World War II10. We propose that the introduction of this exotic population is linked to transport crates, pallets or other military equipment made of untreated lumber from infected trees. The short-range spread of Heterobasidion spp. via infected wood posts has been previously documented 11, but this fungus is generally thought as an unlikely exotic pathogen because of the lack of resting propagules, the short life span of its airborne basidiospores, and its inability to freely grow in the soil. Our findings are noteworthy not only because they indicate fungi such as Heterobasidion may become exotic introductions, but also because they provide new information regarding the poorly studied collateral effects of military operations on natural ecosystems. As in the case of Castelporziano, it may take decades for the effects of introductions to become visible; the time lag between introduction of the disease agent and the development of visible disease symptoms, underlines the urgency to monitor all potential introduction routes.