N. B. Klopfenstein

DNA from bird-dispersed seed and wind-disseminated pollen provides insights into postglacial colonization and population genetic structure of whitebark pine (Pinus albicaulis)

Uniparentally inherited mitochondrial (mt)DNA and chloroplast (cp)DNA microsatellites (cpSSRs) were used to examine population genetic structure and biogeographic patterns of bird‐dispersed seed and wind‐disseminated pollen of whitebark pine (Pinus albicaulis Engelm.). Sampling was conducted from 41 populations throughout the range of the species. Analyses provide evidence for an ancestral haplotype and two derived mtDNA haplotypes with distinct regional distributions. An abrupt contact zone between mtDNA haplotypes in the Cascade Range suggests postglacial biogeographic movements. Among three cpSSR loci, 42 haplotypes were detected within 28 cpSSR sample populations that were aggregated into six regions. Analysis of molecular variance (amova) was used to determine the hierarchical genetic structure of cpSSRs. amova and population pairwise comparisons (FST) of cpSSR, and geographical distribution of mtDNA haplotypes provide insights into historical changes in biogeography. The genetic data suggest that whitebark pine has been intimately tied to climatic change and associated glaciation, which has led to range movements facilitated by seed dispersal by Clark’s nutcracker (Nucifraga columbiana Wilson). The two hypotheses proposed to explain the genetic structure are: (i) a northward expansion into Canada and the northern Cascades in the early Holocene; and (ii) historical gene flow between Idaho and the Oregon Cascades when more continuous habitat existed in Central Oregon during the late Pleistocene. Genetic structure and insights gained from historical seed movements provide a basis on which to develop recovery plans for a species that is at risk from multiple threats.

Effect of white pine blister rust (Cronartium ribicola) and rust-resistance breeding on genetic variation in western white pine (Pinus monticola)

Abstract.Western white pine (Pinus monticola) is an economically and ecologically important species from western North America that has declined over the past several decades mainly due to the introduction of blister rust (Cronartium ribicola) and reduced opportunities for regeneration. Amplified fragment length polymorphism (AFLP) was used to assess the genetic variation in northern Idaho populations of western white pine (including rust-resistant breeding stock) in relation to blister rust. A total of 176 individuals from four populations was analyzed using 163 AFLP loci. Within populations, an average 31.3% of the loci were polymorphic (P), and expected heterozygosity (He) was 0.123. Genetic differentiation values (Gst) showed that 9.4% of detected genetic variation was explained by differences among populations. The comparison between the rust-resistant breeding stock and a corresponding sample derived from multiple natural populations produced similar values of P (35% vs. 34.4%) and He (0.134 vs. 0.131). No apparent signs of a genetic bottleneck caused by rust-resistance breeding were found. However, a comparison of two natural populations from local geographic areas showed that the population with low pressure from blister rust had higher polymorphism and heterozygosity than the population that had experienced high mortality due to blister rust: P (30.7% vs. 25.1%) and He (0.125 vs. 0.100), respectively. In addition, the population from low blister-rust pressure had twice as many unique alleles as the blister rust-selected population. The genetic distance and Dices similarity coefficients among the four populations indicated that the local population that survived high blister-rust pressure was genetically similar to the rust-resistant breeding stock.

Rust disease of eucalypts, caused by Puccinia psidii, did not originate via host jump from guava in Brazil

The rust fungus, Puccinia psidii, is a devastating pathogen of introduced eucalypts (Eucalyptus spp.) in Brazil where it was first observed in 1912. This pathogen is hypothesized to be endemic to South and Central America and to have first infected eucalypts via a host jump from native guava (Psidium guajava). Ten microsatellite markers were used to genotype 148 P. psidii samples from eucalypts and guava plus five additional myrtaceous hosts across a wide geographic range of south‐eastern Brazil and Uruguay. Principal coordinates analysis, a Bayesian clustering analysis and a minimum‐spanning network revealed two major genetic clusters among the sampled isolates, one associated with guava and another associated with eucalypts and three additional hosts. Multilocus genotypes infecting guava differed by multiple mutational steps at eight loci compared with those infecting eucalypts. Approximate Bayesian computation revealed that evolutionary scenarios involving a coalescence event between guava‐ and eucalypt‐associated pathogen populations within the past 1000 years are highly unlikely. None of the analyses supported the hypothesis that eucalypt‐infecting P. psidii in Brazil originated via host jump from guava following the introduction of eucalypts to Brazil approximately 185 years ago. The existence of host‐associated biotypes of P. psidii in Brazil indicates that this diversity must be considered when assessing the invasive threat posed by this pathogen to myrtaceous hosts worldwide.

Proteinase inhibitor II gene in transgenic poplar: Chemical and biological assays

Abstract Transgenic poplar lines were developed to investigate the role of a proteinase inhibitor in pest resistance of woody plants. Using an Agrobacterium binary vector system, the clone ‘Hansen’ ( Populus alba L. × P. grandidentata Michx.) was transformed with chimeric genes containing the coding region of potato proteinase inhibitor II ( PIN 2) linked to either a bacterial nopaline synthase ( nos ) or a cauliflower mosaic virus (35 S ) promoter. All transferred DNA also contained a selectable marker in the form of a nos promoter linked to a neomycin phosphotransferase II ( NPT II ) structural gene. The presence of the transferred PIN 2 and NPT II sequences in poplar was confirmed for nine transgenic lines using polymerase chain reaction (PCR). Expression of PIN 2 in leaves of transgenic poplar was demonstrated by enzyme-linked immunosorbent assays (ELISAs) and western blots. Two unique polypeptides from transgenic poplar, of ca 8 kDa and ca 12 kDa, indicate that PIN 2 was translated appropriately. Resistance to the imported willow leaf beetle was tested in laboratory bioassays. The untransformed clone ‘Hansen’ and 11 transgenic lines were submitted to freshly hatched larvae to determine effects on pupal weight, larval development time and leaf area consumed. A significant difference from the untransformed clone in leaf area consumed was detected in one transgenic line, Tr665. Trends were indicated for several other transgenic lines for the other parameters.

Effects of thidiazuron and benzyladenine on axillary shoot proliferation of three green ash (Fraxinus pennsylvanica Marsh.) clones

Mature seeds of three green ash (Fraxinus pennsylvanica Marsh.) clones, SD1009 (South Dakota origin), SD2002 (South Dakota origin), and KA2018 (Kansas origin) were cut to remove the apical portion and germinated on Murashige and Skoog (1962) salts with B5 vitamins (Gamborg et al., 1968) (MSB5) medium without plant growth regulators. Stable axillary shoot establishment was achieved for all three clones by subculture on MSB5 medium containing a combination of 5 μM thidiazuron (TDZ), 5 μM 6-benzyladenine (BA), and 1 μM indole-3-butyric acid (IBA). Following shoot establishment, axillary shoots were placed on MSB5 medium containing a single treatment of TDZ (1, 5, 10, 20, or 40 μM) or BA (1, 5, 10, 20, 40, or 80 μM). Concentration of TDZ and BA significantly affected shoot biomass (total dry weight of axillary shoots), with 10 μm TDZ or 40 μm BA providing maximum shoot proliferation with all three clones. Significant clonal differences also were noted in the proliferation of axillary shoots, with clone SD1009 exhibiting the highest axillary shoot proliferation. Axillary shoots were rooted under ex vitro conditions and acclimatized to the greenhouse.

Use of flow cytometry, fluorescence microscopy, and PCR- based techniques to assess intraspecific and interspecific matings of Armillaria species

For assessments of intraspecific mating using flow cytometry and fluorescence microscopy, two compatible basidiospore-derived isolates were selected from each of four parental basidiomata of North American Biological Species (NABS) X. The nuclear status in NABS X varied with basidiospore-derived isolates. Nuclei within basidiospore-derived isolates existed as haploids, diploids (doubled haploids), or a mixture of haploids and diploids (doubled haploids). Depending on the nuclear status of the basidiospore-derived lines of NABS X, intraspecifically mated cultures can exist as diploids or tetraploids, and possibly triploids or aneuploids under in vitro conditions. Based on previous in vitro mating studies, seven basidiospore isolates were specifically selected to assess rare, interspecific mating among Armillaria cepistipes, A. sinapina, NABS X, and NABS XI. Cultures from basidiospore-derived isolates were paired to produce four interspecifically paired cultures, and matings were assessed using flow cytometry and restriction fragment length polymorphism (RFLP) analyses. Based on flow cytometric analysis, the A. cepistipes isolate exhibited compatibility with a NABS X isolate, and the A. sinapina isolate exhibited compatibility with a NABS X isolate, and the A. sinapina isolates were individually compatible with isolates of NABS X and NABS XI. Mean fluorescence intensities of A. cepistipes‹NABS X, A. sinapina‹NABS X, and A. sinapina‹NABS XI mated cultures revealed a triploid or tetraploid nuclear status compared to the haploid or diploid (doubled haploid) nuclear status of initial basidiospore-derived isolates. Polymerase chain reaction (PCR) and RFLP of the intergenic spacer (IGS) region generated banding patterns for basidiospore-derived isolates and mated cultures. Four species-specific RFLP banding patterns were observed in basidiospore-derived isolates of A. cepistipes, A. sinapina, NABS X, and NABS XI. PCR-RFLP analysis showed combined banding patterns from mated cultures. Flow cytometry and PCR-RFLP analysis are eective tools to assess matings of Armillaria species.

Genetic diversity and structure of western white pine (Pinus monticola) in North America: a baseline study for conservation, restoration, and addressing impacts of climate change

Western white pine (Pinus monticola) is an economically and ecologically important species in western North America that has declined in prominence over the past several decades, mainly due to the introduction of Cronartium ribicola (cause of white pine blister rust) and reduced opportunities for regeneration. Amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity and structure among populations at 15 sites (e.g., provenances) across the native range of western white pine. The level of genetic diversity was different among 15 populations tested using 66 polymorphic AFLP loci. Nei’s gene diversity (HE) at the population level ranged from 0.187 to 0.316. Genetic differentiation (GST) indicated that 20.1% of detected genetic variation was explained by differences among populations. In general, populations below 45oN latitude exhibited a higher level of genetic diversity than higher latitude populations. Genetic distance analysis revealed two major clades between northern and southern populations, but other well-supported relationships are also apparent within each of the two clades. The complex relationships among populations are likely derived from multiple factors including migration, adaptation, and multiple glacial refugia, especially in higher latitudes. Genetic diversity and structure revealed by this study will aid recognition and selection of western white pine populations for species management and conservation programs, especially in consideration of current and future climate changes.

Assessing host specialization among aecial and telial hosts of the white pine blister rust fungus, Cronartium ribicola

The white-pine blister rust fungus, Cronartium ribicola Fisch. in Rabenh., continues to spread in North Amer- ica, utilizing various aecial (primary) and telial (alternate) hosts, some of which have only recently been discovered. This introduced pathogen has been characterized as having low genetic diversity in North America, yet it has demonstrated a capacity to invade diverse environments. The recent discovery of this rust fungus on the telial host Pedicularis racemosa Dougl. ex Benth., raises questions of whether this host association represents a recent acquisition by C. ribicola or a long- standing host association that was overlooked. Here we explore two questions: (i) is host specialization detectable at a lo- cal scale and (ii) is the capacity to infect Pedicularis racemosa local or widespread? Genetic analysis of C. ribicola iso- lates from different aecial and telial hosts provided no evidence for genetic differentiation and showed similar levels of expected heterozygosity within a geographic population. An inoculation test showed that diverse C. ribicola sources from across North America had the capacity to infect Pedicularis racemosa. These results support a hypothesis that ability to in- fect Pedicularis racemosa is common in C. ribicola from North America. Utilization of Pedicularis racemosa by C. ribicola may be dependent on the co-occurrence of this host, inoculum, and favorable environments.

Integrating concepts of landscape ecology with the molecular biology of forest pathogens

Increasingly more research has focused on characterizing diversity within forest pathogen populations using molecular markers but few studies have characterized features of the landscape that help create or maintain this diversity. Forest diseases commonly occur in patchy distributions across natural landscapes which can be reflected in the genetic composition of the fragmented pathogen populations. This metapopulation structure has seldom been examined by forest pathologists but we believe it offers a potential means to understand the genetic ecology of pathogens in natural landscapes. Molecular markers can be used to detect, identify, and measure detailed differences among subpopulations of forest pathogens. Geographical information systems, spatial analysis and modeling, digital imagery of remotely sensed images, and other tools of landscape ecology provide the means to detect and interpret patterns associated with genotypic asymmetry. Integrating the tools and concepts of molecular biology and landscape ecology by focusing on metapopulation disease phenomena offers a way of conceptually linking molecules and ecosystems.

Tracking down worldwide Puccinia psidii dispersal

Puccinia psidii causes rust disease on many host species in the Myrtaceae [1]. First reported in 1884 on guava in Southern Brazil [2], the rust has since been detected on several myrtaceous in South America, Central America, Caribbean, Mexico, USA: in Florida, California, and Hawaii. More recently, P. psidii was reported in Japan infecting M. polymorpha[3]. Of special note is that a rust was found infecting Myrtaceae species in Australia, where the fungus was reported as Uredo rangelii, based on the tonsure found on the urediniospores surface. However, DNA sequence data did not differentiate that rust from P. psidii[4], and the same tonsure patch, was also observed on rust urediniospores collected from several host species in Brazil [unpublished data].We have hypothesed that P. psidii was introduced into Hawaii through California by trade of rust infected myrtaceous plants, and that P. psidii populations from South America are distinct from the rust populations that became estabilished in California and Hawaii.