Population Genetics of the Ascomycete Phaeocryptopus gaeumannii

by Patrick Bennett, PhD Candidate.  Department of Botany and Plant Pathology at Oregon State University

After finishing my Bachelor of Science degree in Biology at Humboldt State University in 2012, I came to Corvallis, Oregon to pursue a graduate degree with Dr. Jeff Stone in Botany and Plant Pathology at Oregon State University. The Pacific Northwest is a mycological wonderland, and I feel compelled to take full advantage of all of the opportunities that this region has to offer.  Although I have tried to spend as much time as possible in the outdoors, most of my time has been spent in the classroom teaching and learning, and in the laboratory collecting data for my thesis research.  Teaching has been a major part of my responsibilities in grad school.  I have taught a variety of undergraduate and graduate courses ranging from introductory biology to mycology and plant pathology.  I am currently in my 5th (and final!) year as a PhD student and am planning to defend my thesis in June 2018.

My research is primarily focused on the population genetics of the ascomycete Phaeocryptopus gaeumannii.  This is likely the most abundant fungus in western Oregon and Washington, as it is found in nearly every Douglas-fir needle.  Its causes an economically and ecologically important disease known as Swiss needle cast (SNC) by reproducing in the foliage of its host. Its spore-bearing structures (pseudothecia, technically) obstruct stomata, the pores through which plants exchange gases and water vapor with the atmosphere, leading to an inhibition of gas exchange and photosynthesis.  Once the fungus begins to form pseudothecia in a needle, its ability to take in CO2 from the atmosphere is impaired and the foliage cannot produce sugars-the building blocks of complex carbohydrates.  The symptoms of SNC include chlorotic (yellow) foliage, premature shedding of needles, and growth reduction.  It was first recognized in the 1920s in Switzerland (hence the name) and was then reported from Douglas-fir timber plantations across Europe and New Zealand in the following decades.

Subsequent surveys showed that P. gauemannii is likely native to the Pacific Northwest, and although once considered harmless in the native range of Douglas-fir, it is now inflicting devastating economic losses in the region’s Douglas-fir timber industry and causing ecological impacts that are only beginning to be understood.  Prior to the 1990s, outbreaks of SNC only occurred in Christmas tree plantations or where Douglas-fir was planted as an exotic. In the mid 1980s, foresters in coastal northwestern Oregon began to notice the symptoms of the disease, particularly around the town of Tillamook where Douglas-fir trees from various seed sources had been planted in the reforestation efforts after a devastating series of fires that burned over 350,000 acres between 1933 and 1951. By the 1990s, the SNC problem in this region had reached epidemic proportions. This outbreak led to an increase in research efforts to understand its emergence.  The disease now affects approximately 600,000 acres in Oregon and over 300,000 acres in Washington, with estimates of economic impacts around $198 million per year in Oregon alone.

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The fruiting bodies of Phaeocryptopus gaeumannii cause Swiss Needle Cast (SNC) by blocking the stomata (pores used for gas exchange) in Douglas-fir needles. Left: A pseudothecium of P. gaeumannii, showing the asci. Right: The undersides of Douglas-fir needles infected with P. gaeumannii showing pseudothecia protruding from the stomata. Photo credit: Jeff Stone, Oregon State University.

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The effects of SNC can be seen in the growth rings of infected trees. Phaeocryptopus gaeumannii interferes with photosynthesis leading to a decline in the productivity of Douglas-fir trees. Over time, this can affect growth rate. The tree on the left had reduced growth rings, a telltale sign of prolonged SNC infection, while the one on the right was not infected. Photo credit: Jeff Stone, Oregon State University.

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Douglas-fir trees infected with P. gaeumannii exhibit symptoms of Swiss needle cast including chlorosis (yellow-brown foliage), and thin crowns resulting from premature foliage loss. Healthy Douglas-fir trees in the Coast Ranges in Oregon and Washington typically hold 4-5 years of foliage, while trees with severe SNC in this region often only have 1-2 years of foliage remaining. Above: Douglas-fir trees with very low foliage retention. Photo credits: Jeff Stone, Oregon State University.

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On the stand level, SNC appears as a combination of yellow-brown foliage and thinning crowns. The scattered Douglas-fir trees at this site are showing classic symptoms of the disease. Photo credit: Jeff Stone, Oregon State University.

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At the landscape level, SNC appears as patches of Douglas-fir trees with a yellow discoloration compared to the surrounding greenery. These can be measured and quantified in aerial surveys. Photo credit: Rob Flowers, Oregon Department of Forestry, pnwhandbooks.org

Studies of the molecular ecology and population genetics in the late 1990s and early 2000s revealed that what we know as P. gauemannii actually comprises two distinct lineages, or cryptic species (designated Lineage 1 and Lineage 2), that occur with varying abundances across the Oregon Coast Range.  Initial studies of their geographic distributions, and their relationships with SNC disease severity, seemed to implicate Lineage 2 in the recent emergence and intensification of SNC in western Oregon.  Lineage 2 was found in the highest abundance where SNC disease was most severe in the western Coast Range and was absent at sites further inland where SNC was less severe. Sites where Lineage 2 was recovered in the highest abundance seemed to have lower foliage retention and more severe chlorosis.  Lineage 2 was also twice as likely as Lineage 1 to be recovered from severely diseased stands, and only half as likely as Lineage 1 to be recovered from healthy stands.  These discoveries formed the basis of my PhD research.

The factors contributing to the geographic distributions of the two lineages in Oregon, and the relationships between these cryptic species and SNC disease severity are still not well understood.  I began investigating these questions by employing the molecular tools developed by a previous researcher.  The only way to identify a P. gaeumannii isolate as belonging to Lineage 1 or Lineage 2 is by using molecular tools.  There are repeat regions of DNA in the genome known as microsatellites, or short sequence repeats (SSRs), that are highly variable and are unique to individuals, like a DNA “fingerprint.”  These can be used to group individuals into populations because all of the offspring of a given parent share some similarity in these SSRs, and individuals that are not closely related (or have been geographically isolated for long periods of time) are more dissimilar.  Because they are divergent and reproductively isolated, the two lineages of P. gaeumannii differ at these genetic loci (but are otherwise indistinguishable). Our plan was to use the polymerase chain reaction (PCR) to amplify these SSR markers from the genomes of P. gaeumannii isolates from around the world, and analyze them to determine the distributions of these two lineages, examine their population structure (i.e. the distribution of genetic variation in their populations), and quantify genetic diversity in native and introduced populations.

I isolated P. gaeumannii from foliage that was collected across the Oregon and Washington Coast Ranges.  I grew the fungus in culture and purified the DNA, and used PCR to amplify the SSR microsatellite markers.  The PCR products were then genotyped at the Center for Genome Resources and Biocomputing (CGRB) at OSU. The resulting SSR genotypes are still being analyzed, but we have made a few interesting discoveries so far that are described in a peer-reviewed article published in the open-access journal Forests in 2016.  I have now amassed a data set of SSR genotypes from over 3,000 P. gaeumannii isolates collected from the U.S., New Zealand, Australia, and Europe.  Analyses of this data set will be presented as a major part of my PhD thesis in 2018.

Another goal of my research into the evolutionary biology of this fungus is to determine whether the two P. gaeumannii lineages should be considered separate species. Although there are no morphological differences between the two lineages, there is ample evidence to conclude that they are reproductively isolated- enough, by some standards, to call them separate species.  Unfortunately, this “biological species concept” is not adequate in the freaky world of fungi.  The more robust “phylogenetic species concept” is the gold standard for describing new species of fungi. To determine whether the two P. gaeumannii lineages constitute separate species we will need to compare the sequences of several genetic loci between multiple individual isolates.  If the gene sequences from Lineage 1 isolates differ substantially from those of the Lineage 2 isolates, then we could conclude that they are distinct species.  This would lead to major changes in nomenclature (i.e. the names used to describe these fungi) and would give us a better understanding of their evolutionary relationships to other fungi.  We could also combine these data with our microsatellite data set to reconstruct the evolutionary history of this fungus and determine when and where the most recent common ancestor may have existed.  This will provide many insights into the geographic origin of this fungus, which could allow us to identify its center of origin, and identify the regions where Douglas-fir might be most tolerant to P. gaeumannii infection.

This work is motivated by a need to understand the biology and epidemiology of this fungus in order to better mitigate the future impacts of SNC.  This disease is very closely linked with climate, and climate change is likely to exacerbate its effects and expand the area affected by the disease in the coming decades.  Innovations in SNC management will be crucial to maintaining the health of Douglas-fir forests and the viability of the Douglas-fir timber industry in our Pacific Northwest economy.

For more information on SNC including photos and publications, visit the Swiss Needle Cast Cooperative (SNCC) website at www.sncc.forestry.oregonstate.edu.  Our publication in the open-access journal Forests can be found at the following link: http://www.mdpi.com/1999-4907/7/1/14.

Patrick Bennett a recipient of the Ben Woo Scholarship.  Thank you Patrick for writing this article and sharing your work!  PSMS has an established grant program in honor of its first president Benjamin Woo.  Ben personified the PSMS mission to foster the understanding of Mycology as a science and a hobby. As an organization of volunteer amateur mycologists, PSMS wishes to fund the projects of enthusiasts who need financial assistance to reach their goal related to the study of mycology.  To learn more about the Ben Woo Scholarship, visit psms.org/scholarship.