George Newcombe

Melampsora ‹columbiana, a natural hybrid of M. medusae and M. occidentalis

Hybrids of Melampsora medusae and M. occidentalis are herein described as M. xcolumbiana. This hybrid taxon is characterized by isolates that generally exhibit morphological intermediacy in uredinial and telial traits, and mixed virulence/avirulence on Populus trichocarpa and P. deltoides, the natural hosts of M. occidentalis and M. medusae, respectively. Hybrids frequently contain ribosomal ITS sequences from both Melampsora species, but discordant isolates were also found, like M. medusae in having a urediniospore equatorial smooth spot, and like M. occidentalis in spine density, and intermediate in urediniospore length. Isolates with hybrid urediniospore morphology but homozygous for ITS sequences from M. medusae or M. occidentalis were also found, suggesting that the primary F1 hybrids have produced F2 and/or backcross progeny. In a 1997 survey of the leaf rust population on hybrid poplar in the Pacific Northwest, this ‘new population’ of Melampsora xcolumbiana was the only taxon found. Specimens from nearly a century ago indicate that an older hybrid population is of wide distribution in North America. This ‘older population’ of Melampsora xcolumbiana was found on specimens of all species of Populus in sections Aigeiros and Tacamahaca, and on many of their hybrids, in areas outside the type localities of M. occidentalis and M. medusae. These three Melampsora taxa, M. medusae, M. occidentalis, and M. xcolumbiana, are the only ones that occur naturally on sections Aigeiros and Tacamahaca of Populus in North America.

Hidden diversity of endophytic fungi in an invasive plant

Fungal endophytes are important in plant ecology and common in plants. We attempted to test cointroduction and host-jumping hypotheses on a community basis by comparing endophytes isolated from invasive spotted knapweed (Centaurea stoebe, Asteraceae) in its native and invaded ranges. Of 92 combined, sequence-based haplotypes representing eight classes of Fungi, 78 occurred in only one of the two ranges. In the native range of C. stoebe, one haplotype of Alternaria alternata was clearly dominant, whereas in the invaded range, no haplotype was dominant. Many haplotypes were closely related to one another and novel. For example, six putative, new species of Botrytis were discovered as endophytes of C. stoebe, which has never been reported to have Botrytis spp.. Apparent differences between the two communities of endophytes were significant according to an analysis of similarity, but phylogenetic community structure did not differ significantly between the ranges. Both host-jumping and cointroduction of fungal endophytes likely took place during the spotted knapweed invasion.

Suppressed recombination around the MXC3 locus, a major gene for resistance to poplar leaf rust

Abstract A positional cloning strategy is being implemented in Populus for the isolation of the dominant MXC3 allele, which confers resistance to poplar leaf rust caused by Melampsora×columbiana (pathotype 3). AFLP markers were used to saturate the chromosomal region around the MXC3 locus in a large (n=1,902) Populus trichocarpa×P. deltoides (T×D) mapping pedigree segregating 1:1 for rust resistance and susceptibility. The high-resolution linkage map developed around the MXC3 locus contains 19 AFLP markers and spans a genetic distance of 2.73 cM. Of the 19 AFLP markers, seven were found to co-segregate with the locus. One co-segregating AFLP marker, CCG.GCT_01, was converted to an STS marker (BVS1) and used to identify a physical contig of overlapping BAC clones from the MXC3 region. Genetic and physical mapping of markers isolated from the BAC contig failed to delimit the MXC3 locus within a 300-kb interval defined by the overlapping BAC clones. This result indicates a >25-fold reduction in recombination frequency in the MXC3 region compared to the average rate of recombination for the Populus genome.

Fungal endophytes directly increase the competitive effects of an invasive forb

Competitive outcomes among plants can vary in different abiotic and biotic conditions. Here we tested the effects of two phylotypes of Alternaria endophytes on the growth, competitive effects, and competitive responses of the exotic invasive forb Centaurea stoebe. Centaurea stoebe was a better competitor against North American grass species than grasses from its European home range in the absence of endophytes. However, one endophyte both increased the biomass of C. stoebe and reduced the competitive effect of North American grasses on C. stoebe. The competitive effects of C. stoebe on grass species native to North America were enhanced by both fungal endophytes, but not for native European grasses. We do not know the mechanism by which endophytes increased C. stoebes competitive ability, and particularly against biogeographically new neighbors, but one endophyte increased the competitive ability of C. stoebe without increasing its size, suggesting mechanisms unrelated to increased growth. We tested only a fraction of the different endophytic fungi that have been found in C. stoebe, only scratching the surface of understanding their indirect effects. However, our results are the first to demonstrate such effects of a fungal endophyte infecting an invasive forb, and one of the few to show that endophyte effects on competition do not have to be mediated through herbivory.

Abundant pathogenic variation in the new hybrid rust Melampsora ×columbiana on hybrid poplar.

ABSTRACT The recently described rust hybrid Melampsora xcolumbiana was discovered as a result of its novel pathogenic variation on Populus trichocarpa x P. deltoides (TxD) hybrid poplar. To characterize this pathogenic variation, 10 commercial TxD clones, all F(1) clones, were chosen as host differentials. Fourteen mononuredinial isolates of Pacific Northwestern field collections of M. xcolumbiana, from 1996 to 1998 inclusive, were determined to be 13 distinct pathotypes. In contrast, four Southeastern isolates of M. medusae could not be distinguished on the same TxD host differentials, although they can be distinguished as pathotypes using P. deltoides differentials. The first three pathotypes of M. xcolumbiana (Mxc1, Mxc2, and Mxc3) and a Mississippi isolate of M. medusae were inoculated onto a three-generation TxD pedigree, formerly used to characterize the Mmd1 gene for resistance to M. medusae. Resistance to the Mxc3 pathotype mapped to the same linkage group (group Q) as the Mmd1 gene. In contrast, linked genes for resistance to Mxc1 and to Mxc2 were located on linkage group O, and were unlike Mmd1 and Mxc3 in that they were inherited from P. deltoides. The latter two genes resembled Mmd1 and Mxc3 in that infection type was correlated with quantitative traits such as uredinial density and latent period. Pathogenic variation in M. xcolumbiana matches resistance genes from both P. trichocarpa and P. deltoides and reveals the vulnerability to hybrid rust of commercial TxD hybrid poplar clones.

Endophytes influence protection and growth of an invasive plant

We investigated the symbiotic activities of fungal endophytes isolated from spotted knapweed, Centaurea stoebe. Previously, an analysis of community similarity had demonstrated differences in the endophyte communities of C. stoebe in its native and invaded ranges. Here, we found that specific endophytes can exert positive effects on their host, whereas others exert negative effects. Endophytes produced metabolites that inhibited germination of a competitor of C. stoebe. Endophytes also repelled a specialist insect herbivore, perhaps by producing biologically active volatiles. Yet other endophytes acted as cryptic pathogens of C. stoebe, suppressing its germination, reducing its growth, increasing the abundance of a generalist insect herbivore, and delaying or suppressing its flowering. Since, as reported here, endophytes are not functionally interchangeable, previously reported community differences could be contributing to the invasiveness of C. stoebe.

Molecular and morphological characterization of the willow rust fungus, Melampsora epitea, from arctic and temperate hosts in North America.

Current taxonomy places all rust fungi that occur on willow (Salix spp.) in North America in one species complex, Melampsora epitea Thüm. Characteristics of M. epitea isolates from the Canadian arctic were compared to M. epitea isolates from temperate regions of North America. Sequences from internal transcribed spacer (ITS) regions of rDNA were obtained from urediniospores from rust-infected Salix leaves collected in the Canadian arctic and in Minnesota and compared. Phylogenetic analysis of nuclear ribosomal ITS regions indicated that arctic M. epitea samples were divergent from temperate M. epitea isolates, perhaps in part because all rusts examined diverged according to host species. Four urediniospore characteristics were examined: area, circularity (shape factor), major axis length and spine density. Statistically significant (P < 0.05) differences were observed for spine density among all host species except S. nigra and S. bebbiana. However major axis length differed between these species. These results represent the first evidence that arctic and temperate Melampsora species on Salix hosts in North America have evolved distinct molecular and morphological characters.

A novel plant-fungal mutualism associated with fire.

Bromus tectorum, or cheatgrass, is native to Eurasia and widely invasive in western North America. By late spring, this annual plant has dispersed its seed and died; its aboveground biomass then becomes fine fuel that burns as frequently as once every 3-5 y in its invaded range. Cheatgrass has proven to be better adapted to fire there than many competing plants, but the contribution of its fungal symbionts to this adaptation had not previously been studied. In sampling cheatgrass endophytes, many fire-associated fungi were found, including Morchella in three western states (New Mexico, Idaho, and Washington). In greenhouse experiments, a New Mexico isolate of Morchella increased both the biomass and fecundity of its local cheatgrass population, thus simultaneously increasing both the probability of fire and survival of that event, via more fuel and a greater, belowground seed bank, respectively. Re-isolation efforts proved that Morchella could infect cheatgrass roots in a non-mycorrhizal manner and then grow up into aboveground tissues. The same Morchella isolate also increased survival of seed exposed to heat typical of that which develops in the seed bank during a cheatgrass fire. Phylogenetic analysis of Eurasian and North American Morchella revealed that this fire-associated mutualism was evolutionarily novel, in that cheatgrass isolates belonged to two phylogenetically distinct species, or phylotypes, designated Mel-6 and Mel-12 whose evolutionary origin appears to be within western North America. Mutualisms with fire-associated fungi may be contributing to the cheatgrass invasion of western North America.

Fungal endophyte increases the allelopathic effects of an invasive forb.

Abstract Endophytic plant symbionts can have powerful effects on the way their hosts interact with pathogens, competitors, and consumers. The presence of endophytes in plants can alter food webs, community composition and ecosystem processes, suggesting that endophyte-plant symbioses may represent unique forms of extended phenotypes. We tested the impact of the fungal endophyte Alternaria alternata (phylotype CID 120) on the allelopathic effect of the invasive forb Centaurea stoebe when in competition with the North American native bunchgrass Koeleria macrantha in a greenhouse competition experiment. The allelopathic effect of C. stoebe on K. macrantha when infected with the fungal endophyte was more than twice that of endophyte-free C. stoebe. However, this allelopathic effect was a small part of the very large competitive effect of C. stoebe on K. macrantha in all treatments, likely because of the priority effects in our experimental design. To our knowledge, these results are the first experimental evidence for a symbiotic relationship between plants and fungal endophytes affecting allelopathic interactions between competing plants, and thus provide insight into the mechanisms by which fungal endophytes may increase the competitive ability of their hosts.

Leaf endophytes and Populus genotype affect severity of damage from the necrotrophic leaf pathogen, Drepanopeziza populi

Fungal leaf endophytes—nonpathogenic microfungi that live within plant leaves—are ubiquitous in land plants. Leaf endophytes and host plant genotypes may interact to determine plant disease severity. In a greenhouse inoculation experiment, we found that leaf endophyte species and Populus angustifolia genotypes both affected disease outcomes in plants inoculated with the necrotrophic leaf pathogen Drepanopeziza populi. Contrary to many studies showing endophytes conferring defense, all plant genotypes inoculated with the endophyte Penicillium sp. prior to inoculation with the pathogen D. populi were characterized by greater pathogen symptom severity than plants inoculated with the pathogen only. We quantified defense gene expression via qRT–PCR, but found no evidence that increased pathogen damage was related to differential expression of the assayed genes. A second endophyte, Truncatella angustata, which was previously found to reduce symptom severity of the biotrophic pathogen Melampsora in Populus trichocarpa, did not affect symptom severity of the necrotrophic pathogen D. populi or defense gene expression. Overall, our study highlights the variable effects of endophytes on pathogen symptom severity, and illustrates that plant genotypic variation can remain important for disease outcomes even in the presence of endophytes altering disease. Additional work is needed to elucidate the mechanism by which fungal leaf endophytes alter disease in their host plants.