Ned B. Klopfenstein

Molecular Characterization of Fusarium oxysporum and Fusarium commune Isolates from a Conifer Nursery

ABSTRACT Fusarium species can cause severe root disease and damping-off in conifer nurseries. Fusarium inoculum is commonly found in most container and bareroot nurseries on healthy and diseased seedlings, in nursery soils, and on conifer seeds. Isolates of Fusarium spp. can differ in virulence; however, virulence and colony morphology are not correlated. Forty-one isolates of Fusarium spp., morphologically indistinguishable from F. oxysporum, were collected from nursery samples (soils, healthy seedlings, and diseased seedlings). These isolates were characterized by amplified fragment length polymorphism (AFLP) and DNA sequencing of nuclear rDNA (internal transcribed spacer including 5.8S rDNA), mitochon-drial rDNA (small subunit [mtSSU]), and nuclear translation elongation factor 1-alpha. Each isolate had a unique AFLP phenotype. Out of 121 loci, 111 (92%) were polymorphic; 30 alleles were unique to only highly virulent isolates and 33 alleles were unique to only isolates nonpathogenic on conifers. Maximum parsimony and Bayesian analyses of DNA sequences from all three regions and the combined data set showed that all highly virulent isolates clearly separated into a common clade that contained F. commune, which was recently distinguished from its sister taxon, F. oxysporum. Interestingly, all but one of the nonpathogenic isolates grouped into a common clade and were genetically similar to F. oxysporum. The AFLP cladograms had similar topologies when compared with the DNA-based phylograms. Although all tested isolates were morphologically indistinguishable from F. oxysporum based on currently available monographs, some morphological traits can be plastic and unreliable for identification of Fusarium spp. We consider the highly virulent isolates to be F. commune based on strong genetic evidence. To our knowledge, this is the first reported evidence that shows F. commune is a cause of Fusarium disease (root rot and dampingoff) on Douglas-fir seedlings. Furthermore, several AFLP genetic markers and mtSSU sequences offer potential for development of molecular markers that could be used to detect and distinguish isolates of F. oxysporum nonpathogenic to conifers and highly virulent isolates of F. commune in forest nurseries.

Approaches to predicting potential impacts of climate change on forest disease: an example with Armillaria root disease

Climate change will likely have dramatic impacts on forest health because many forest trees could become maladapted to climate. Furthermore, climate change will have additional impacts on forest health through changes in the distribution and severity of forest disease. Methods are needed to predict the influence of climate change on forest disease so that appropriate forest management practices can be implemented to minimize disease impacts. Initial approaches for predicting the future distribution of pathogens are dependent on reliable data sets that document the current, precise location of accurately identified pathogens and hosts. Precise distribution information can be used in conjunction with available climate surfaces to determine which climatic factors and interactions influence species distribution. This information can be used to develop bioclimatic models to predict the probability of suitable climate space for host and pathogen species across the landscape. A similar approach using climate surfaces under predicted future climate scenarios can be used to project suitable climate space for hosts and pathogens in the future. Currently such predictions are well developed for many forest host species, but predictive capacity is extremely limited for forest pathogens because of lacking distribution data. Continued surveys and research are needed to further refine bioclimatic models to predict influences of climate and climate change on forest disease.

Influence of Host Resistance on the Genetic Structure of the White Pine Blister Rust Fungus in the Western United States

Cronartium ribicola, the causal agent of white pine blister rust, has been devastating to five-needled white pines in North America since its introduction nearly a century ago. However, dynamic and complex interactions occur among C. ribicola, five-needled white pines, and the environment. To examine potential evolutionary influences on genetic structure and diversity of C. ribicola in western United States, population genetic analyses of C. ribicola were conducted using amplified fragment length polymorphism (AFLP) molecular markers. The fungus was sampled at six sites. Collections for two of the six sites were from separate plantings of resistant-selected western white pine and sugar pine. Heterozygosity based on polymorphic loci among populations ranged from 0.28 to 0.40, with resistant-selected plantations at the extremes. Genetic differentiation was also highest between these two populations. Principal coordinates analysis and Bayesian assignment placed most isolates that are putative carriers of virulence to major-gene resistance into a discernable cluster, while other isolates showed no clustering by site or host species. These results indicate that C. ribicola in western North America is not genetically uniform, despite its presumed single site of introduction and relatively brief residence. Moreover, major-gene resistance appears to have imposed strong selection on the rust, resulting in reduced genetic diversity. In contrast, no evidence of selection was observed in C. ribicola from hosts that exhibit only multigenic resistance.

Insights into the phylogeny of Northern Hemisphere Armillaria: Neighbor-net and Bayesian analyses of translation elongation factor 1-α gene sequences

ABSTRACT Armillaria possesses several intriguing characteristics that have inspired wide interest in understanding phylogenetic relationships within and among species of this genus. Nuclear ribosomal DNA sequence–based analyses of Armillaria provide only limited information for phylogenetic studies among widely divergent taxa. More recent studies have shown that translation elongation factor 1-α (tef1) sequences are highly informative for phylogenetic analysis of Armillaria species within diverse global regions. This study used Neighbor-net and coalescence-based Bayesian analyses to examine phylogenetic relationships of newly determined and existing tef1 sequences derived from diverse Armillaria species from across the Northern Hemisphere, with Southern Hemisphere Armillaria species included for reference. Based on the Bayesian analysis of tef1 sequences, Armillaria species from the Northern Hemisphere are generally contained within the following four superclades, which are named according to the specific epithet of the most frequently cited species within the superclade: (i) Socialis/Tabescens (exannulate) superclade including Eurasian A. ectypa, North American A. socialis (A. tabescens), and Eurasian A. socialis (A. tabescens) clades; (ii) Mellea superclade including undescribed annulate North American Armillaria sp. (Mexico) and four separate clades of A. mellea (Europe and Iran, eastern Asia, and two groups from North America); (iii) Gallica superclade including Armillaria Nag E (Japan), multiple clades of A. gallica (Asia and Europe), A. calvescens (eastern North America), A. cepistipes (North America), A. altimontana (western USA), A. nabsnona (North America and Japan), and at least two A. gallica clades (North America); and (iv) Solidipes/Ostoyae superclade including two A. solidipes/ostoyae clades (North America), A. gemina (eastern USA), A. solidipes/ostoyae (Eurasia), A. cepistipes (Europe and Japan), A. sinapina (North America and Japan), and A. borealis (Eurasia) clade 2. Of note is that A. borealis (Eurasia) clade 1 appears basal to the Solidipes/Ostoyae and Gallica superclades. The Neighbor-net analysis showed similar phylogenetic relationships. This study further demonstrates the utility of tef1 for global phylogenetic studies of Armillaria species and provides critical insights into multiple taxonomic issues that warrant further study.

Armillaria mexicana, a newly described species from Mexico

ABSTRACT Armillaria mexicana (Agaricales, Physalacriaceae) is described as a new species based on morphology, DNA sequence data, and phylogenetic analyses. It clearly differs from previously reported Armillaria species in North, Central, and South America. It is characterized by the absence of fibulae in the basidioma, abundant cheilocystidia, and ellipsoidal, hyaline basidiospores that are apparently smooth under light microscope, but slightly to moderately rugulose under scanning electron microscope. It is differentiated from other Armillaria species by macromorphological characters, including annulus structure, pileus and stipe coloration, and other structures. DNA sequence data (nuc rDNA internal transcribed spacers [ITS1-5.8S-ITS2 = ITS], 28S D-domain, 3′ end of 28S intergenic spacer 1, and translation elongation factor 1-α [TEF1]) show that A. mexicana sequences are quite distinct from sequences of analogous Armillaria species in GenBank. In addition, sequences of ITS of the A. mexicana ex-type culture reveal an ITS1 of 1299 bp and an ITS2 of 582 bp, the longest ITS regions reported thus far in fungi. Phylogenetic analysis based on TEF1 sequences place A. mexicana in a well-separated, monophyletic clade basal to the polyphyletic A. mellea complex.

Root Diseases in Coniferous Forests of the Inland West : Potential Implications of Fuels Treatments

After nearly 100 years of fire exclusion, introduced pests, and selective harvesting, a change in forest composition has occurred in many Inland West forests of North America. This change in forest structure has frequently been accompanied by increases in root diseases and/or an unprecedented buildup of fuels. Consequently, many forest managers are implementing plans for fuels treatments to lower the risk of severe wildfires. Impacts on root disease should be considered before selecting appropriate fuels treatments. Complex interactions exist among conifer root diseases, fuels treatments, forest structure, species composition, stand history, and other environmental factors. As forest managers prescribe fuels treatments, their success in lowering the risk of severe wildfire will depend in part on the impacts of these treatments on root disease. Root diseases are one of many factors to be considered when developing plans for fuels treatments. Choices must be made on a site-by-site basis, with knowledge of the diseases that are present. This paper provides examples of how fuels treatments may increase or reduce specific diseases and demonstrates their importance as considerations in the fuels management planning process. Several root diseases prevalent within Inland West of North America are addressed: Armillaria root disease, annosus root disease, laminated root rot, black stain root disease, Schweinitzii root and butt rot, Tomentosus root disease, Rhizina root rot, and stringy butt rot. For each disease, general information is provided on disease identification, management options, and potential effects of fuels treatments. However, many long-term studies are needed to assess effects of specific interactions among fuels treatments, root diseases, and host trees.

Molecular Genetic Approaches Toward Understanding Forest-Associated Fungi and Their Interactive Roles Within Forest Ecosystems

Purpose of ReviewThe continued, rapid development of novel molecular genetic tools is contributing to a better understanding of forest-associated fungi and their interactive roles within diverse forest ecosystems. This paper focuses on recent developments of DNA-based diagnostics/detection, phylogenetics, population genetics, genomics, and metagenomics tools that have been applied to forest-associated fungi to better understand their roles in forest ecosystems and provide key insights for managing forest health.Recent FindingsWith the advent of new molecular technologies, we can better understand the biology of forest fungi by examining their genetic code. By utilizing genomics, fungal pathogens’ biological functions can be deduced from its genomic content. Further, high-resolution marker systems allow the determination of a pathogen’s population genetics and genomics, which provides important insights into its global movement and genetic shifts in local pathogen populations. Such genetic information has diverse applications for forest management to improve forest health. Lastly, new technologies in metagenomics will enhance the abilities to detect, describe, and utilize the complex interactions among fungal pathogens/symbionts, host trees, and associated microbial communities to develop novel management strategies for forest ecosystems.SummaryContinued development and applications of molecular genetic and genomic tools provide insights into the diverse roles of forest-associated fungi in forest ecosystems, but long-term, wide-scale research is needed to determine how ecological functions are influenced by complex ecological interactions among microbial communities, other forest ecosystem components, and the environment. Such approaches may foster a paradigm shift away from single microbial pathogens, decomposers, or symbionts interacting with a single host or substrate, and provide more holistic approaches toward understanding interactions among microbial communities that drive forest health processes.