A genetic analysis of 151 isolates of the Sudden Oak Death pathogen provides new evidence that the commercial plant trade possibly helped introduce the microbe to the United States, according to plant pathologists at the University of California, Berkeley.
The study, in the May issue of the journal Molecular Ecology and published online on Thursday, April 13, has potentially shed light on how Phytophthora ramorum - the pathogen responsible for Sudden Oak Death - a contagious, fungus-like disease that has killed hundreds of thousands of oaks and tanoaks along the Pacific Coast - may have been unwittingly introduced into the United States.
In the study, a single lineage was found in isolates taken from U.S. forests, and a second distinct lineage was found in samples from European nurseries. It was determined that the isolates from U.S. forests could be traced back to a single individual strain.
While both lineages had been identified previously, the discovery of a new, third lineage in U.S. nurseries was a surprise to the researchers.
The third lineage of P. ramorum was present, along with the other two known lineages, in nurseries from Washington state.
"We found a lot of genetic information and traits within this third lineage that are different from the other two," said Matteo Garbelotto, UC Berkeley associate extension specialist and adjunct professor, and principal investigator of the study. "So, this species must have a lot more diversity than we thought originally."
The authors indicate that because native pathogens are characterized by great genetic diversity, the lack of such diversity in U.S. forests shows that the disease was introduced.
"This is the strongest evidence yet to support the view that this pathogen was introduced onto our continent via international plant trade," said Kelly Ivors, lead author of the paper. Ivors was a UC Berkeley post-doctoral research associate in ecosystem sciences at the time of the study and is now an assistant professor in plant pathology at North Carolina State University.
Since it takes millions of years for a distinct lineage to develop, and this organism is not endemic in the United States, the researchers concluded that the known lineages in this country's nurseries are the result of the introduction of at least three strains rather than the result of changes that occurred after the organism was introduced.
"The big surprise was that in U.S. nurseries, we find not only both lineages from European nurseries and U.S. forests, but also a third lineage that was not known before this study," said Garbelotto. "So, to date, all the diversity that is known in this species has been found in some U.S. nurseries. Furthermore, we also found in some West Coast nurseries a genotype identical to the one that caused the epidemic in forests. This study provides evidence that the pathogen was introduced to forested areas on the West Coast and suggests that U.S. nurseries may have been a stepping stone for the introduction."
In 2004, the commercial plant trade found itself in an unwelcome spotlight when two Southern California nurseries inadvertently shipped infected plants nationwide to more than 20 states. To date, the pathogen, originally discovered in California wildlands in 1995, now is known to have more than 100 susceptible host plants and has been detected in nurseries or retail garden stores in 21 U.S. states, in British Columbia and throughout Europe. Nursery plants found positive for P. ramorum are quickly destroyed.
Currently in this country, wildland, watershed and soil surveys outside of California and Oregon have all been negative for the pathogen. But it may take years for symptom expression to be identifiable in a natural setting, so years of monitoring are necessary to consider an area absent of disease.
The researchers pointed out that the previously unknown third lineage was either discovered in or traced back to nurseries in Washington state. Because all three known lineages are present in that state, Garbelotto said an understanding of trading routes for nursery plants leading to Washington may help reveal the exact origin of the pathogen.
The researchers analyzed 71 isolates of P. ramorum from the United States and 80 isolates from nine countries within the European Union. The U.S. isolates were taken from 12 counties in California, two in Oregon and one in Washington. The samples, tested at UC Berkeley and Plant Research International in the Netherlands, represent the known geographic range of the pathogen and its hosts.
The P. ramorum genome was sequenced by the U.S. Department of Energy in 2004, enabling the researchers of this study to use a process called microsatellite analysis to determine the genetic variability of the isolates. They identified 12 independent markers on the genome and looked for variations among them. Because the markers are neutral - they do not code for any function or trait - any variations would be independent of each other and not related to selection pressures.
P. ramorum and many other plant pathogens, in general, are capable of both sexual and asexual - or clonal - reproduction. Chemical signals are sent out to attract the opposite sex, but if the other mating type is absent, the organism switches to clonal reproduction.
"The information we have from the P. ramorum genome indicates that in the pathogen's areas of origin, sexual reproduction must be occurring," said Garbelotto. "If the pathogen is introduced, we typically only find one mating type which spreads by cloning itself. This kind of self-cloning that we found in P. ramorum has been suggested by previous studies and is now fully confirmed by results from this study."
He said the lack of variation or a slight variation limited to one marker indicates that the sample is a clone, and that a great deal of variability in the markers signals a sample that is the result of sexual recombination. "The results of our microsatellite analysis not only tell us that we have three groups, or lineages, that are genetically different from one another, but that within the lineage found in U.S. forests, there is not a lot of diversity," said Garbelotto. "That indicates that the pathogen is not spreading through sexual reproduction in the U.S. forests."
In addition to lineages, the researchers looked at genetic diversity among the isolates. They recovered 10 of 11 genotypes - or 91 percent - from the nursery isolates compared with only two genotypes - 18 percent - in samples taken from the wild throughout California and Oregon. The authors suggest that the higher genotypic diversity in nurseries may be due to the repeated exchange of pathogen genotypes through the trade of infected plant material, from natural mutations of introduced genotypes occurring in nurseries, or both.
"It doesn't appear as if mating is currently occurring, but new, potentially more harmful strains could develop in the United States if strains from different lineages were to intermix and mate," said Garbelotto. "Not only do we need to avoid the spread of new strains to uninfected areas, but we also need to avoid spreading new lineages in areas where one lineage may already be present. Researchers are working hard to determine all factors that influence pathogen dispersal and containment. Until answers are available, the USDA regulations are helping to prevent further spread."
Other authors of the paper include Noah Rosenzweig, a post-doctoral researcher at UC Berkeley; and Peter Bonants, senior scientist at Plant Research International.
Funding for this research was provided by the Gordon and Betty Moore Foundation, the USDA Forest Service-Pacific Southwest Research Station, and the Dutch Plant Protection Service.
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