Colonization history, host distribution, anthropogenic influence and landscape features shape populations of white pine blister rust, an invasive alien tree pathogen.

White pine blister rust is caused by the fungal pathogen Cronartium ribicola J.C. Fisch (Basidiomycota, Pucciniales). This invasive alien pathogen was introduced into North America at the beginning of the 20th century on pine seedlings imported from Europe and has caused serious economic and ecological impacts. In this study, we applied a population and landscape genetics approach to understand the patterns of introduction and colonization as well as population structure and migration of C. ribicola. We characterized 1,292 samples of C. ribicola from 66 geographic locations in North America using single nucleotide polymorphisms (SNPs) and evaluated the effect of landscape features, host distribution, and colonization history on the structure of these pathogen populations. We identified eastern and western genetic populations in North America that are strongly differentiated. Genetic diversity is two to five times higher in eastern populations than in western ones, which can be explained by the repeated accidental introductions of the pathogen into northeastern North America compared with a single documented introduction into western North America. These distinct genetic populations are maintained by a barrier to gene flow that corresponds to a region where host connectivity is interrupted. Furthermore, additional cryptic spatial differentiation was identified in western populations. This differentiation corresponds to landscape features, such as mountain ranges, and also to host connectivity. We also detected genetic differentiation between the pathogen populations in natural stands and plantations, an indication that anthropogenic movement of this pathogen still takes place. These results highlight the importance of monitoring this invasive alien tree pathogen to prevent admixture of eastern and western populations where different pathogen races occur.

First Report of Rust Caused by Puccinia psidii on Paperbark, Melaleuca quinquenervia, in California.

Infections by Puccinia psidii Winter were detected on Melaleuca quinquenervia (Cav.) Blake (paperbark, tea tree) during a regular inspection of a San Diego, California nursery in November 2010 by San Diego County inspectors. Urediniospore morphology, production of bright-yellow uredinia on both upper and lower leaf surfaces, DNA sequencing, and inoculation tests confirm the identity of the pathogen. From digital image analysis, dimensions of 49 urediniospores from the paperbark rust collection were 25 (19 to 30) × 21 (18 to 23) µm, typical of uredinia of P. psidii (3). Some urediniospores also had a distinctive, unornamented "tonsure" near the base, and all pustules lacked teliospores. Both latter features are considered by some (3) as more typical of an asexual sub-taxon, Uredo rangelii Simpson, Tho., Grgur. which however, has narrower urediniospores than we report. U. rangelii is also considered less of a threat to Eucalyptus than other variants of P. psidii in the broad sense (1). The 627-bp DNA sequence of the internal transcribed spacer (ITS) region of the paperbark rust (GenBank Accession No. JF960255) obtained by John Hanna at the USDA Forest Service, Forestry Sciences Lab, Moscow, ID using PCR and ITS1/ITS4 primers had over 99% identity with 18 GenBank sequences of P. psidii from diverse Myrtaceae, including Melaleuca, Psidium, and Eucalyptus. In January and February of 2011, inoculations of 7- to 10-cm cuttings of active terminal growth demonstrated pathogenicity to the three species of Myrtaceae that were tested: paperbark, common myrtle Myrtus communis L., and brush cherry Syzygium australe (J.C. Wendl. ex Link) B. Hyland (=Eugenia australis, =E. myrtifolia) cv. Monterey Bay. Uredinia developed within 10 days of inoculation on inoculated host cuttings. Proportions of cuttings infected for paperbark, common myrtle, and brush cherry were 5 of 7, 6 of 6, and 1 of 6, respectively; pustules were most abundant on common myrtle and least abundant (a single pustule) on brush cherry. Similarly treated control cuttings were not infected. P. psidii has previously caused severe outbreaks on paperbark in Florida (2). California interceptions and nursery detections of P. psidii are recorded in the California Department of Food and Agriculture's internal, unpublished, Pest Detection Database, have all occurred since 2003, and include Florida sources. From the Database, detections on materials shipped into California nurseries have been: brush cherry from Florida, March and April, 2004 at Valley Center, CA; allspice (Pimenta dioica (L) Merr.) from Florida, June 2005 at Gilroy, CA; and Java apple/rose apple (Syzygium samarangense [Blume] Merr. & L.M. Perry [= Eugenia javanica Lam.]) from Hawaii, 2008 at Anaheim, CA. Other California nursery infestations have been: brush cherry, April 2004, at Valley Center, CA; pohutukawa (Metrosideros excelsa Solander ex J. Gaertner), July 2007 at Bonsall, CA; and common myrtle, November 2008 at Fallbrook, CA. The rust has not been reported in California on any plants outside of nursery settings or on Eucalyptus in any setting. The current strains of P. psidii in California appear likely to remain a recurrent problem for ornamental plant and foliage crops in the myrtle family grown in some nursery settings. References: (1) A. J. Carnegie et al. Australas. Plant Pathol. 39:463, 2010. (2) M. B. Rayachhetry et al. Biol. Control 22:38, 2001. (3) J. A. Simpson et al. Australas. Plant Pathology 35:549, 2006.

First Report of Bot Canker Caused by Diplodia corticola on Coast Live Oak (Quercus agrifolia) in California.

Sharp decline and mortality of coast live oak (Quercus agrifolia) has been observed in San Diego County, CA since 2002. Much of this decline has been attributed to a new pest in California, the goldspotted oak borer (GSOB, Agrilus coxalis) (1). Symptoms include crown thinning, bark cracking and/or peeling, patches of stain (1 to 10 cm in diameter), bleeding on the bole, and tree death and are most often observed on trees with a diameter at breast height (DBH) >30 cm. In 2008, a Botryosphaeria sp. was recovered from necrotic tissue of bleeding bole cankers from GSOB-affected trees in Jamul, CA. Zone lines separated dead and live tissue in affected phloem and xylem. Pycnidia were observed on the bark surface of the infected host. Fifty conidia averaging 32 × 18 µm, one-septate with age, and morphologically similar to conidia described by Úrbez-Torres et al. were observed (4). Oak stands with tree mortality were surveyed in GSOB-infested and -uninfested sites over eight locations throughout San Diego and Riverside counties in 2009 and 2010. Symptomatic tissue or conidia from pycnidia of affected trees, plated onto potato dextrose agar amended with 0.01% tetracycline and incubated at 25°C for 1 week, consistently produced cultures with dense, wooly, olive-green mycelium. Mycelia fit the description of Botryosphaeria corticola A.J.L. Phillips, Alves et Luque (anamorph Diplodia corticola) (2). The resulting amplified ITS4/5 region of two sequences matched 100% to published D. corticola sequences (GU799472 and GU799460) (4). These sequences were deposited with NCBI GenBank (HM104176 and HM104177). Koch's postulates were conducted by inoculating 2-mm-diameter holes on five coast live oak trees with D. corticola. Holes were drilled to the cambium at 2 to 4 locations per tree within 1 to 2 m up the bole using a 0.157-cm portable electric drill. Trees ranged from 3.7- to 32.4-cm DBH. Either single agar plugs from two isolates each of a 7-day-old culture (UCR454 and UCR793) or noncolonized agar plugs as uninoculated controls were inserted into the holes and then covered with petroleum jelly and Parafilm. Average temperature was 10°C, relative humidity of 64%, and no precipitation during inoculation. Inoculations were conducted at a location in San Diego County uninfested by GSOB and repeated twice. After 3.5 months, bark was removed from inoculation sites. Average lesion length was not significantly different between inoculations, thus data were combined (one way analysis of variance [ANOVA]; P = 0.05). Lesions averaged 13.9 × 2.3 cm and were significantly different (n = 30; one way ANOVA; P = 0.05) from controls that measured 0.31 × 0.3 cm. Staining was observed around the inoculation points on all trees and three trees exhibited bleeding. Necrotic tissue was observed in the phloem and 3 mm into the xylem tissue, where the lesion had extended up and down the grain. D. corticola was consistently reisolated from necrotic tissue but not from control treatments. B. corticola was originally described as a canker pathogen on Quercus spp. in the western Mediterranean (2), and is known to contribute to the decline of cork oak (Q. suber) in the region (3). To our knowledge, this is the first report of D. corticola causing bot canker on coast live oak in California. References: (1) T. W. Coleman and S. J. Seybold. U. S. For. Serv. R5-PR-08, 2008. (2) A. Correia et al. Mycologia 96:598, 2004. (3) J. Luque et al. For. Pathol. 38:147, 2008. (4) J. R. Úrbez-Torres et al. Plant Dis. 94:785, 2010.

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 White Pine Blister Rust Fungus, Cronartium ribicola, on Pedicularis bracteosa.

Until recently, Cronartium ribicola J.C. Fisch. was thought to utilize only Ribes spp. (Grossulariaceae) as telial hosts in North America. During 2004, Pedicularis racemosa Dougl. ex Benth. and Castilleja miniata Dougl. (Orobanchaceae) were proven as natural telial hosts at a subalpine site (48.634109°N, 116.570817°W, elevation 1,800 m) near Roman Nose Lake, ID, where whitebark pine (Pinus albicaulis Engelm.) and western white pine (Pinus monticola Dougl. ex D. Don) are aecial hosts, and Pedicularis, Castilleja, and Ribes spp. are common herbs/shrubs (2). During August 2006, teliospore columns typical of C. ribicola or the morphologically indistinguishable (2) C. coleosporioides J.C. Arthur were found on two Pedicularis bracteosa Benth. plants at this site, within 3 m of a large, sporulating canker on whitebark pine. ITS/5.8S rDNA regions were sequenced using detached teliospore column samples from the two plants, ITS1F and ITS4 primers (3), and standard PCR protocols (2). One sample sequence was identified as C. ribicola and the other as C. coleosporioides (GenBank Accession Nos. EF185857 and EF185858, respectively), by exact matches in comparisons with published sequences (2). Artificial inoculation confirmed P. bracteosa's ability to host C. ribicola. Sections of leaves collected near Freezeout Saddle, ID (47.00885°N, 116.00846°W, elev. 1,600 m) were rinsed in water, placed abaxial side up on moistened filter paper in 150-mm petri plates, inoculated with seven diverse sources of urediniospores/aeciospores, misted with distilled water, and incubated at 18°C with 12 h of light. A single leaf section produced urediniospores 17 days and teliospores 26 days after inoculation with one of two Roman Nose aeciospore sources. Urediniospores from this leaf section caused infections on Ribes nigrum L., and teliospore columns yielded a DNA sequence that matched C. ribicola. Though P. bracteosa is confirmed as yet another natural host of C. ribicola in North America, it may be producing less C. ribicola inoculum for pine infection than do the P. racemosa and Ribes spp. telial hosts at the collection site. Uredinia and telia of C. ribicola on P. bracteosa were much less frequent and smaller than those on P. racemosa and Ribes spp. and those of C. coleosporioides on this same host (2). Pedicularis (but not Castilleja) spp. are significant telial hosts of C. ribicola strains at some high elevation sites in eastern Asia (1). Discovery of multiple North American telial hosts in the Orobanchaceae suggests unrecognized complexity in C. ribicola's ability to exploit ecological niches in recently established pathosystems of North America (2). References: (1) G. I. McDonald et al. Pages 41-57 in: Forest Pathology: From Genes to Landscapes. J. Lundquist and R. Hamelin, eds. The American Phytopathological Society. St. Paul, MN, 2005. (2) G. I. McDonald et al. For. Pathol. 36:73, 2006. (3) T. J. White et al. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al. eds. Academic Press, San Diego, CA, 1990.

Gene Action and Linkage of Avirulence Genes to DNA Markers in the Rust Fungus Puccinia graminis.

ABSTRACT Two strains of the wheat stem rust fungus, Puccinia graminis f. sp. tritici, were crossed on barberry, and a single F(1) progeny strain was selfed. The parents, F(1), and 81 F(2) progeny were examined for virulence phenotypes on wheat differential cultivars carrying stem rust resistance (Sr) genes. For eight Sr differentials, phenotypic ratios are suggestive of single dominant avirulence genes AvrT6, AvrT8a, AvrT9a, AvrT10, AvrT21, AvrT28, AvrT30, and AvrTU. Avirulence on the Sr; (Sr 'fleck') differential showed phenotypic ratios of approximately 15:1, indicating epistatic interaction of two genes dominant for avirulence. Avirulence on Sr9d favored a 3:13 over a 1:3 ratio, possibly indicating two segregating genes-one dominant for avirulence and one dominant for avirulence inhibition. Linkage analysis of eight single dominant avirulence genes and 970 DNA markers identified DNA markers linked to each of these avirulence genes. The closest linkages between AvrT genes and DNA markers were between AvrT6 and the random amplified polymorphic DNA marker crl34-155 (6 centimorgans [cM]) AvrT8a and the amplified fragment length polymorphism marker eAC/mCT-197 (6 cM) and between AvrT9a and the amplified fragment length polymorphism marker eAC/mCT-184 (6 cM). AvrT10 and AvrTU are linked at distance of 9 cM.

Variation at the b Mating Type Locus of Ustilago maydis.

ABSTRACT Population level diversity at the Ustilago maydis b mating type locus was determined in samples from four Minnesota locations using a combination of plate mating techniques and a polymerase chain reaction (PCR)-based assay. The PCR method allows rapid identification of b types from samples of natural populations and utilizes the hypervariable regions of the b locus that determine mating type specificity. Results demonstrated high levels of b diversity within populations, with one population yielding 17 of the total 18 b types found in the study. Pairwise G(ST) values were in the range of 0.02 to 0.05, and common b mating types were found across broad geographic distances. These data demonstrated that very low levels of differentiation among U. maydis populations occur with respect to b locus variation. Consistent with frequency-dependent selection models, b types were represented at approximately equal frequencies within the entire Minnesota population. However, neutral evolutionary models for patterns of geographic distribution and variation at b cannot be entirely excluded. The importance to agricultural practices of understanding population genetic processes is discussed.