Fusarioid community diversity associated with conifer seedlings in forest nurseries across the contiguous USA.

Introduction: Fusarioid fungi that cause damping-off and root diseases can result in significant losses to conifer crops produced in forest nurseries across the USA. These nurseries are vital to reforestation and forest restoration efforts. Understanding the diversity of Fusarioid fungi associated with damping-off and root diseases of conifer seedlings can provide an approach for targeted management techniques to limit seedling losses and pathogen spread to novel landscapes. Methods: This study identifies 26 Fusarium spp. (F. acuminatum, F. annulatum, F. avenaceum, F. brachygibbosum, F. clavus, F. commune, F. cugenangense, F. diversisporum, F. elaeagni, F. elaeidis, F. flocciferum, F. fredkrugeri, F. fujikuroi, F. grosmichelii, F. ipomoeae, F. lactis, F. languescens, F. luffae, F. odoratissimum, F. oxysporum, F. queenslandicum, F. redolens, F. torulosum, F. triseptatum, F. vanleeuwenii, & F. verticillioides), 15 potential species within Fusarium and Neocosmospora species complexes (two from F. fujikuroi species complex, nine from F. oxysporum species complex, three from F. tricinctum species complex, and one from Neocosmospora species complex), and four Neocosmospora spp. (N. falciforme, N. metavorans, N. pisi, & N. solani) and associated host information collected from conifer-producing nurseries across the contiguous USA. Results: Phylogenetic analyses identified Fusarioid fungi haplotypes that were associated with 1) host specificity, 2) localization to geographic regions, or 3) generalists found on multiple hosts across diverse geographic regions. Discussion: The haplotypes and novel species identified on conifer seedlings should be considered for further analysis to determine pathogenicity, pathogen spread, and assess management practices.

Genetic diversity and population structure of Raffaelea quercus-mongolicae, a fungus associated with oak mortality in South Korea.

Raffaelea quercus-mongolicae is a fungus associated with oak wilt and deemed to cause extensive oak mortality in South Korea. Since the discovery of this fungus on a dead Mongolian oak (Quercus mongolica) in 2004, the mortality continued to spread southwards in South Korea. Despite continued expansion of the disease and associated significant impacts on forest ecosystems, information is lacking about the origin and genetic diversity of R. quercus-mongolicae. Restriction-site-Associated DNA (RAD) sequencing was used to assess genetic diversity and population structure among five populations (provinces) of R. quercus-mongolicae in South Korea. In total, 179 single nucleotide polymorphisms (SNPs) were identified among 2,639 RAD loci across the nuclear genome of the 54 R. quercus-mongolicae isolates (0.0012 SNPs per bp), which displayed an overall low expected heterozygosity and no apparent population structure. The low genetic diversity and no apparent population structure among South Korean populations of this ambrosia beetle-vectored fungus supports the hypothesis that this fungus was introduced to South Korea.

First Report of the Armillaria Root-Disease Pathogen, Armillaria gallica, Associated with Several Woody Hosts in Three States of Mexico.

In September 2007, rhizomorphs with morphological characteristics of Armillaria were collected from woody hosts in forests of Mexico State, Veracruz, and Oaxaca, Mexico. Based on pairing tests, isolates were assigned to five somatically compatible genets or clones (MEX7R, MEX11R, MEX23R, MEX28R, and MEX30R). These genets were all identified as Armillaria gallica based on somatic pairing tests against known tester isolates and nucleotide sequences of the translation elongation factor 1α (tef-1α; GenBank Accession Nos. KF156772 to 76). Sequences of tef-1α for all genets showed a max identity of 97 to 99% to A. gallica (ST23, JF313125) (3,4). However, A. gallica comprises a genetically diverse complex that likely represents multiple cryptic species (3). In Mexico, this species has been previously reported in northeastern Morelos on Quercus sp., eastern Mexico State on Pinus hartwegii, and southwestern Mexico State on Prunus persica (1,2). This study identified associations with 10 new hosts within three states of Mexico, but only five hosts were diseased. Genet MEX7R comprised seven isolates collected in the University of Chapingo forest near Texcoco, Mexico State (19°18'10.764″ N, 98°42'14.147″ W, elevation 3441 m). Four MEX7R isolates were collected from diseased Alnus sp. including the root ball of a 130 cm dbh, root-disease killed tree, one isolate from a symptomless Senecio sp. s.l. (Roldana sp.) shrub and two isolates from symptomless Abies religiosa. Genet MEX11R comprised four isolates from a cloud forest near Xalapa, Veracruz (19°31'14.628″ N, 96°59'22.812″ W, elevation 1496 m). MEX11R isolates were collected from the roots of a root-disease killed Carpinus caroliniana, and from trees with no obvious symptoms (Miconia mexicana, Quercus xalapensis, and Liquidambar styraciflua). Two isolates of genet MEX23R were collected from the Jardin Botanico Francisco Javier Clavijero, Instituto de Ecologia, A.C., Xalapa, Veracruz (19°30'49.067″ N, 96°56'32.999″ W, elevation 1344 m). These isolates were from root-diseased Eriobotrya japonica (non-native fruit tree) that showed obvious symptoms (flaccid, chlorotic, and senescing leaves) and from an adjacent, infected Platanus mexicana that did not show readily observable symptoms. Two collections near Oaxaca, Oaxaca, included a single isolate MEX28R from the root ball of a recently root disease-killed Arbutus xalapensis within a small root disease center at Peña Prieta, in Parque La Cumbre, near Ixtepeji (17°09'42.084″ N, 96°38'15.936″ W, elevation 2853 m) and a single isolate MEX30R from the base of an asymptomatic Alnus acuminata near the El Carrizal fish hatchery 10 km northeast of San Miguel del Valle (17°06'45.036″ N, 96°24'03.743″ W, elevation 2594 m). Armillaria gallica has a circumpolar distribution with an extremely wide host range, and its ecological behavior varies greatly. Continued surveys are needed to better understand the distribution and ecological impacts of this pathogen in relation to Armillaria root disease in Mexico and the potential influences of climate change. Although A. gallica displays diverse ecological behavior, trees infected with A. gallica are less likely to survive the stresses of human activity and a changing climate (4). References: (1) D. Alvarado-Rosales and R. A. Blanchette. Phytopathology 84:1106, 1994. (2) R. D. Elias-Roman et al. For. Pathol. 43:390, 2013. (3) M.-S. Kim et al. Phytopathology 102:S4.63, 2012. (4) B. Marcais and N. Breda. J. Ecol. 94:1214, 2006.

First Report of the Armillaria Root Disease Pathogen, Armillaria gallica, on Douglas-fir (Pseudotsuga menziesii) in Arizona.

In August 2010, a mycelial fan (isolate AZ32F) of Armillaria sp. was collected from the root collar of a living Douglas-fir tree on the Mogollon Rim within the Coconino National Forest (approximate location 34°25'31.26″N, 111°20'41.04″W, elevation 2,293 m) in central Arizona. Mycelial fans under the bark of living trees are a sign of pathogenicity, and symptoms of the diseased tree included resinosis, sloughing bark, and thinning crown. The infected tree was located on a south-facing slope with approximately 30% tree cover, dominated by ponderosa pine (Pinus ponderosa), with lesser components of Douglas-fir and Gambel oak (Quercus gambelii). Based on three replications of somatic incompatibility tests against 24 tester isolates representing seven North American Armillaria spp., isolate AZ32F showed 100% intraspecific compatibility (colorless antagonism) with all four A. gallica isolates, 22% compatibility with A. calvescens, and 0% compatibility with the remaining Armillaria spp. Based on GenBank BLASTn of isolate AZ32F sequences, the partial LSU-IGS1 (GenBank Accession No. KF186682) showed 99 to 100% similarity to A. gallica and two other related Armillaria spp. with 99 to 100% coverage, and translation elongation factor-1 alpha (tef-1α) sequences (KC525954) showed 96% similarity to A. gallica (JF895844) with 100% coverage. Thus, isolate AZ32F was identified as A. gallica, based on somatic incompatibility tests and DNA sequences (partial LSU-IGS1 and tef-1α). Although the isolate is identified as A. gallica with similarities to other North American isolates, evidence is mounting that currently recognized A. gallica likely represents a species complex that comprises multiple phylogenetic species (4). Previous surveys in Arizona have noted A. mellea and A. solidipes (as A. ostoyae) (3), but A. gallica has never been previously confirmed in this state. Within North America, A. gallica is commonly reported east of the Rocky Mountains and in West Coast states of the United States, where it infects hardwoods and conifers including Douglas-fir (1,2). Its ecological behavior ranges from saprophyte to weak/aggressive pathogen (1,2). Because damage by A. gallica appears to increase on hosts predisposed by stress (1), further surveys are needed to document its distribution, frequency, and ecological behavior in the southwestern United States, where climate change will likely cause tree stress due to maladaptation. Continued surveys for Armillaria spp. will better determine their potential threat within the geologically and ecologically unique Mogollon Rim of Arizona. References: (1) K. Baumgartner and D. M. Rizzo. Plant Dis. 85:947, 2001. (2) N. J. Brazee and R. L. Wick. For. Ecol. Manage. 258:1605, 2009. (3) R. L. Gilbertson and D. M. Bigelow. J. Arizona-Nevada Acad. Sci. 31:13, 1998. (4) M.-S. Kim et al. Phytopathology 102:S4.63, 2012.

First Report of Puccinia psidii Caused Rust Disease Epiphytotic on the Invasive Shrub Rhodomyrtus tomentosa in Florida.

Rhodomyrtus tomentosa (Aiton) Hassk. (downy-rose myrtle, family: Myrtaceae), of South Asian origin, is an invasive shrub that has formed monotypic stands in Florida (3). During the winter and spring of 2010 through 2012, a rust disease of epiphytotic proportion was observed on young foliage, stem terminals, and immature fruits of this shrub in natural areas of Martin and Lee counties, Florida. Expanding leaves and succulent stems developed chlorotic flecks on the surface that developed into pustules and ruptured to discharge urediniospores. Symptomatic leaves and stems developed severe necrotic spots and resulted in tissue distortion, defoliation, and stem dieback. Based on symptoms and urediniospore morphology and dimensions (17.7 to 26.1 [22.1 ± 0.3] × 14.7 to 21.1 [17.7 ± 0.2] µm; n = 51) (4), the causal agent was identified as Puccinia psidii Winter; teliospores were not observed in samples since it does not produce these spore stages below 20°C ambient temperature (1). This identification was confirmed by a GenBank BLAST of internal transcribed spacer (ITS) sequences (Accession Nos. KC607876 and KC607877) that showed 99% identity with 42 sequences of P. psidii from diverse host species and locations. P. psidii is believed to be of neotropical origin and has a host range of 129 species in 33 genera within Myrtaceae (2). However, P. psidii caused disease of downy-rose myrtle has not been previously reported in Florida, even though severe infections occurred on another invasive tree, Melaleuca quinquenervia (Cav.) S.F. Blake (3), growing in adjacent areas. In December 2011, urediniospores were collected from downy-rose myrtle, established in aqueous suspension (45,000 spores/ml), and spray inoculated on potted downy-rose myrtle plants (n = 3), which were maintained in 100% ambient humidity, at 20°C, with a 12-h light cycle for 72 h. Plants mock-inoculated with water served as the negative control. Disease symptoms, including chlorotic flecks and raised surfaces, appeared on leaf lamina in 3 to 6 days on P. psidii-inoculated plants, while control plants remained symptomless. Raised surfaces developed into distinct pustules and eventually erupted to discharge urediniospores within 6 to 12 days of inoculation. Tests were repeated once during March and April of 2012 with the same results. The latent and incubation periods reported herein are within the previously reported range for P. psidii (2,4). To our knowledge, this is the first confirmed report of P. psidii epiphytotic on downy-rose myrtle populations in Florida. The recent occurrence of P. psidii epiphytotic on downy-rose myrtle raises critical questions as to why this myrtle rust disease is so severe and widespread on this host after decades of presumed exposure to P. psidii in Florida. Because this rust pathogen has emerged as a major invasive threat to many myrtaceous species around the world, further genotyping and cross-inoculation studies are needed to determine the host specificity and potential origin of the P. psidii isolates derived from downy-rose myrtle (2). References: (1) A. C. Alfenas et al. Australas. Plant Pathol. 32:325, 2003. (2) A. J. Carnegie and J. R. Lidbetter. Australas. Plant Pathol. 41:13, 2012. (3) K. A. Langeland and C. K Burks, eds. Identification and biology of non-native plants in Florida's natural areas. University of Florida, Gainesville, 1998. (4) M. B. Rayachhetry et al. Biol. Contr. 22:38, 2001.

First Report of an Armillaria Root Disease Pathogen, Armillaria gallica, Associated with Several New Hosts in Hawaii.

The loss and decline of native tree species caused by invasive plant pathogens is a major threat to the endangered endemic forests of the Hawaiian Islands (3). Thus, it is critical to characterize existing pathogens to evaluate potential invasiveness. In August 2005, rhizomorphs and mycelial bark fans of genet HI-4 were collected from dead/declining, mature trees of introduced Monterey pine (Pinus radiata) on the southern flank of Mauna Kea, Hawaii (approximately 19°42'55″N, 155°26'48″W, elevation 2,175 m). In March of 2008, three additional genets (HI-11, HI-13, and HI-16) were collected as rhizomorphs at a site named Pu'u La'au (west slope of the Mauna Kea Forest Reserve area, approximately 19°50'00″N, 155°35'35″W, elevation 2,275 to 2,550 m), approximately 20 km west-northwest of the HI-4 collection. These genets were collected from apparently healthy loblolly pine (Pinus taeda) that were introduced, apparently healthy mamane (Sophora chrysophylla; an endemic tree species of Hawaii), dead and dying mamane, and apparently healthy Methley plum (Prunus cerasifera × Prunus salicina) that was planted. All isolates were determined to have identical sequences in the intergenic spacer-1 rDNA region (GenBank Accession No. DQ995357). On the basis of somatic paring tests against North American Armillaria tester strains and 99% nucleotide sequence identities to GenBank Accession Nos. AY190245 and AY190246, these isolates were identified as Armillaria gallica. Past surveys have noted A. mellea sensu lato and A. nabsnona on numerous hosts in Hawaii, including mamane (3,4). However, to our knowledge, this is the first confirmed report of A. gallica in Hawaii, where it was found on mamane, Monterey pine, loblolly pine, and Methley plum. A. gallica has been widely categorized as a beneficial saprophyte, an opportunistic pathogen, or an aggressive pathogen (2). A recent study suggests that A. gallica can be highly pathogenic in some areas of the eastern United States and it is an important component of forest decline (2), especially under increasing stressors such as climate change. The isolation of A. gallica from declining stands on both introduced and endemic hosts under drought conditions suggests this pathogen is a contributing factor to forest decline on the island of Hawaii. Because the mamane tree is an important component of the native forest stands and essential to the endangered palila bird (Loxioides bailleui), which feeds almost exclusively on its green seeds (1), continued monitoring of Armillaria root disease is warranted. References: (1) P. C. Banko et al. J. Chem. Ecol. 28:1393, 2002. (2) N. J. Brazee and R. L. Wick. For. Ecol. Manage. 258:1605, 2009. (3) R. E. Burgan and R. E. Nelson. USDA For. Serv. Tech. Rep. PSW-3, 1972. (4) J. W. Hanna et al. Plant Dis. 91:634, 2007.

First Report of Armillaria Root Disease Caused by Armillaria tabescens on Araucaria araucana in Veracruz, Mexico.

In September 2007, bark samples were collected from the root collar of a single Araucaria araucana tree that had recently died and was suspected of being killed by Armillaria root disease. Disease symptoms and signs included a thinning crown and fruiting bodies at the tree base over a several-year period before tree death. The tree was located in an isolated street-tree planting within a business district on Maestros Veracruzanos Street, Xalapa, Veracruz (19°31'52''N, 96°54'25''W, elevation 1,392 m). One fungal isolate (MEX21WF) was obtained, which possessed two sequence repeat types from the intergenic spacer-1 (IGS-1) region (GenBank Accession Nos. GQ335541 and GQ335542). On the basis of these IGS-1 sequences, this isolate from Mexico possessed 99% nucleotide sequence identities with North American Armillaria tabescens isolates (GenBank Accession Nos. AY695410 ≈ GQ335541 and AY773966 ≈ GQ335542). Somatic pairing tests of the isolate with other North American Armillaria species also identified it as A. tabescens (2). In addition, fruiting bodies were produced on the stump base in 2009 that matched morphological features of A. tabescens, e.g., exannulate, cespitose growth in clusters, brown-gray stipe to blackish toward the base, longitudinally fibrillose, basidiospores (6-) 7 to 9 × 4 to 5 (-5.5) µm, and other general morphology. On the basis of these three lines of taxonomic evidence, it was concluded that the isolate was A. tabescens. To our knowledge, this is the first confirmed report of A. tabescens causing Armillaria root disease in Mexico. Furthermore, this note represents the first report of A. tabescens on Araucaria araucana, which is native to Chile and Argentina. The other previous reports of A. tabescens in Mexico are based on herbarium specimens collected in 1965 (BPI 753040) from Valle de Bravo (approximately 350 km west of Xalapa) in the state of México and 1973 (BPI 753041) from near Monterrey (approximately 760 km north-northwest of Xalapa) in the state of Nuevo León (1). However, no host information or confirmation of taxonomic identification was reported for these herbarium specimens. Although this note confirms the presence of A. tabescens in Mexico, more surveys and monitoring are needed to determine the full distribution of this pathogen in Mexico. Because the climate and tree communities of eastern Mexico are similar to those of the southeastern United States, where A. tabescens has been reported as a common pathogen of oaks and fruit trees (3,4), it seems reasonable that A. tabescens may represent an existing or potential threat in eastern Mexico. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory. Online publication. ARS, USDA, 2010. (2) K. I. Mallett and Y. Hiratsuka. Can. J. Bot. 64:2588, 1986. (3) F. Miranda and A. J. Sharp. Ecology 31:313, 1950. (4) G. Schnabel et al. Mycol. Res. 109:1208, 2005.

First Report of Armillaria sinapina, a Cause of Armillaria Root Disease, Associated with a Variety of Forest Tree Hosts on Sites with Diverse Climates in Alaska.

In August of 2007, a preliminary survey was conducted in Alaska to evaluate potential impacts of climate change on forest trees. Armillaria sinapina, a root-disease pathogen, was isolated from conifer and hardwood hosts on climatically diverse sites spanning 675 km from the Kenai Peninsula to the Arctic Circle. Seven isolates (NKAK1, NKAK2, NKAK5, NKAK6, NKAK9F, NKAK13, and NKAK15) were identified as A. sinapina by using intergenic spacer-1 nucleotide sequences (GenBank Accession Nos. EU665175-EU665181) and somatic pairings. Of particular note is that one isolate (NKAK9F) was obtained from a declining Salix sp. (willow) growing in a flood plain near the Arctic Circle (66°32.316'N, 150°47.717'W). This isolate was collected from mycelial bark fans that were intercalated within multiple bark layers, a sign of disease. All other isolates were derived from rhizomorphs attached to and/or embedded within roots and root collars, but most host trees showed no clear indication of disease. Two isolates were collected from dead trees within a small mortality center (62°08.703'N, 150°04.593'W) that included an isolate from Picea glauca (white spruce; NKAK13) and another isolate from Betula sp. (birch; NKAK15). Additional isolates came from a beetle-killed P. glauca (NKAK1) 120 km northwest of Anchorage (61°48.079'N, 148°16.983'W) and a suppressed (overtopped by other trees in the stand) Tsuga mertensiana (mountain hemlock; NKAK2) 58 km southeast of Anchorage (60°50.679'N, 149°03.742'W). The two remaining isolates originated from the Kenai Peninsula (approximately 60°29.629'N, 149°45.465'W) and were derived from a root-diseased Populus tremuloides (trembling aspen; NKAK5) and a suppressed P. glauca (NKAK6). Although A. mellea sensu lato was previously reported on willow in interior Alaska (1) and A. sinapina was previously reported from sites under coastal influence (4), this represents the first confirmed report of A. sinapina on P. glauca, T. mertensiana, Populus tremuloides, Salix sp., and Betula sp. in Alaska. Unfortunately, pathogenicity of A. sinapina cannot be readily verified under experimental conditions because environmental variables, host-tree status (e.g., species, population, age, and vigor), and inoculum potential are difficult to recreate. Armillaria sinapina is typically regarded as a weak pathogen of diverse hosts (3). However, A. sinapina is predicted to cause more disease on hosts predisposed by climate stress, and climate change is well-documented in Alaska (2). Because A. sinapina occurs on diverse hosts under different climates across a wide geographic range in Alaska, Armillaria root disease could become more prevalent on trees stressed by climate change. References: (1) T. E. Hinds and T. H. Laurent. Plant Dis. Rep. 62:972, 1978. (2) J. J. McCarthy et al., eds. Climate Change 2001: Impacts, Adaptation and Vulnerability. Cambridge University Press, Cambridge, 2001. (3) D. J. Morrison et al. Can. J. Plant Pathol. 7:242, 1985. (4) C. G. Shaw, III and E. M. Loopstra. Phytopathology 78:9714, 1988.

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.

First Report of the Root-Rot Pathogen, Armillaria nabsnona, from Hawaii.

The genus Armillaria (2) and Armillaria mellea sensu lato (3) have been reported previously from Hawaii. However, Armillaria species in Hawaii have not been previously identified by DNA sequences, compatibility tests, or other methods that distinguish currently recognized taxa. In August 2005, Armillaria rhizomorphs and mycelial bark fans were collected from two locations on the island of Hawaii. Stands in which isolates were collected showed moderate to heavy tree mortality and mycelial bark fans. Pairing tests (4) to determine vegetative compatibility groups revealed three Armillaria genets (HI-1, HI-7, and HI-9). Rhizomorphs of genet HI-1 were collected from both dead and healthy mature trees of the native 'Ohia Lehua (Metrosideros polymorpha) approximately 27 km west of Hilo, HI (approximately 19°40'49″N, 155°19'24″W, elevation 1,450 m). Rhizomorphs of HI-7 and HI-9 were collected, respectively, from dead/declining, mature, introduced Nepalese alder (Alnus nepalensis) and from an apparently healthy, mature, introduced Chinese banyan (Ficus microcarpa) in the Waipi'o Valley (approximately 20°03'29″N, 155°37'35″W, elevation 925 m). On the basis of somatic pairing tests and intergenic spacer-1 (IGS-1) nucleotide sequence identities of 99 to 100% with North American A. nabsnona (GenBank Accession No. AY509178), HI-1 (GenBank Accession No. DQ995356), HI-7 (GenBank Accession No. DQ995358), and HI-9 (GenBank Accession No. DQ995359) were identified as A. nabsnona, a pathogen of hardwoods (1). The IGS-1 sequences of A. nabsnona genets (HI-1, HI-7, and HI-9) had a greater similarity to North American collections of A. nabsnona than to the Asian A. nabsnona, even though the two introduced hosts originated from Asia. Phylogeographic studies could help determine the potential introduction and original source of A. nabsnona in Hawaii. Although A. nabsona was isolated from multiple hosts in declining stands, pathogenicity studies are needed to confirm whether this pathogen causes disease on diverse native and exotic tree species in Hawaii. References: (1) E. Allen et al. Pages 2-7 in: Common Tree Diseases of British Columbia. Natural Resources Canada. Canadian Forest Service, Victoria, BC, Canada, 1998. (2) D. E. Hemmes and D. E. Desjardin. Pages 129 and 153 in: Mushrooms of Hawaii. Ten Speed Press, Berkeley, CA, 2002. (3) F. F. Laemmlen and R. V. Bega. Plant Dis. Rep. 58:102, 1974. (4) Y. Wu et al. USDA Forest Service Tech. Rep. R2-58, 1996.

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

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 (H(e)) was 0.123. Genetic differentiation values (G(st)) 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 H(e) (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 H(e) (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 Dice's 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.

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.

Bulked Segregant Analysis Identifies Molecular Markers Linked to Melampsora medusae Resistance in Populus deltoides.

ABSTRACT In the north central United States, leaf rust caused by Melampsora medusae is a major disease problem on Populus deltoides. In this study we identified molecular markers linked to a M. medusae resistance locus (Lrd1) that was segregating 1:1 within an intraspecific P. deltoides family (C9425DD). Previous field results were confirmed in the controlled environment of a growth chamber through an excised whole-leaf inoculation method. Using bulked segregant analysis we identified two random amplified polymorphic DNA (RAPD) markers (OPG10(340) and OPZ19(1800)) that are linked to Lrd1. Based on segregation in a total of 116 progeny, the genetic distances between OPG10(340) and OPZ19(1800) and the resistance locus were estimated as 2.6 and 7.4 Haldane centimorgans (cM), respectively. Multipoint linkage analyses strongly suggest the most likely order for these loci is Lrd1, OPG10(340), and OPZ19(1800). These markers may prove to be instrumental in the eventual cloning of Lrd1, as well as for marker-assisted selection of leaf-rust resistant genotypes.