A culture-independent approach to understanding the role of soil fungal communities in Bromus tectorum stand failure.

Cheatgrass (Bromus tectorum L.) is an invasive annual grass (Poaceae) that has colonized large portions of the Intermountain West. Cheatgrass stand failures have been observed throughout the invaded region, the cause of which may be related to the presence of several species of pathogenic fungi in the soil or surface litter. In this metabarcoding study, we compared the fungal communities between sites that have and have not experienced stand failure. Samples were taken from the soil and surface litter near Winnemucca, Nevada, and in Skull Valley, Utah. Our results show distinct fungal communities associated with stand failure based on both geography and sample type. In both the Winnemucca and Skull Valley surface litter, there was an elevated abundance of the endophyte Ramimonilia apicalis in samples that had experienced a stand failure. Winnemucca surface litter stand failure samples had an increased abundance of a potential pathogen in the genus Comoclathris. Skull Valley surface litter stand failure samples had an increased abundance of an undescribed new species in the Rustroemiaceae family which is responsible for the so-called bleach blonde syndrome in cheatgrass, while the soils had an increased abundance of potential pathogens in the genera Olpidium and Monosporascus.

Infection of the Native California Grass, Bromus carinatus, by Fusarium circinatum, the Cause of Pitch Canker in Pines.

At Point Reyes National Seashore in California, Fusarium circinatum, the causal agent of pitch canker in pines, was isolated from Pinus muricata, the California native grass, Bromus carinatus, and the introduced grass, Holcus lanatus. All grass plants from which F. circinatum was isolated were symptomless. Pathogenicity of grass isolates was confirmed by inoculation of P. radiata trees, which developed symptoms similar to trees inoculated with a pine isolate of F. circinatum. Isolates from grasses were somatically compatible with isolates recovered from symptomatic pines. B. carinatus grown in a growth chamber was inoculated with a green fluorescent protein-expressing strain of F. circinatum. Segments of inoculated leaves were incubated in moist chambers; after 1 to 2 days, sporulating hyphae were observed growing from leaf tissue. Spores of F. circinatum removed from B. carinatus leaves were confirmed to be fluorescent when illuminated with ultraviolet light. These results raise the possibility that B. carinatus cryptically infected by F. circinatum may be a source of propagules capable of infecting pines.

Phytotoxic Activity of Metabolites Isolated from Rutstroemia sp.n., the Causal Agent of Bleach Blonde Syndrome on Cheatgrass (Bromus tectorum).

A fungal pathogen soon to be described as Rutstroemia capillus-albis (Rutstroemiaceae, Helotiales, Leotiomycetes) has been identified as the causal agent of 'bleach blonde syndrome' on the invasive annual grass weed Bromus tectorum (cheatgrass) in western North America. This apparently common but previously undescribed disease causes premature senescence and sterility, but does not affect seed germination or seedling emergence and growth. This study investigated whether the new species produces phytotoxins that could be implicated in pathogenesis. The compounds 9-O-methylfusarubin, 9-O-methylbostrycoidin, 5-O-methylnectriafurone, trans-methyl-p-coumarate and terpestacin were isolated from the solid culture of this fungus. The undescribed absolute stereochemistry at C-3 of 9-O-methylfusarubin and at C-1' of 5-O-methylnectriafurone were assigned by applying electronic and vibrational circular dichroism (ECD and VCD) combined with computational methods and the advanced Mosher's method, respectively. The first three listed compounds are naphtoquinone pigments, while terpestacin is a sesterterpene, and trans-methyl-p-coumarate could be the product of an unusual fungal phenylpropanoid biosynthesis pathway. In a juvenile plant immersion bioassay, both 9-O-methylfusarubin and terpestacin proved to be highly toxic at 10-4 M, causing wilting and plant death within 10 days. This finding suggests that these two compounds could play a role in pathogenesis on B. tectorum.

Archaea and bacteria mediate the effects of native species root loss on fungi during plant invasion.

Although invasive plants can drive ecosystem change, little is known about the directional nature of belowground interactions between invasive plants, native roots, bacteria, archaea and fungi. We used detailed bioinformatics and a recently developed root assay on soils collected in fescue grassland along a gradient of smooth brome (Bromus inermis Leyss) invasion to examine the links between smooth brome shoot litter and root, archaea, bacteria and fungal communities. We examined (1) aboveground versus belowground influences of smooth brome on soil microbial communities, (2) the importance of direct versus microbe-mediated impacts of plants on soil fungal communities, and (3) the web of roots, shoots, archaea, bacteria and fungi interactions across the A and B soil horizons in invaded and non-invaded sites. Archaea and bacteria influenced fungal composition, but not vice versa, as indicated by redundancy analyses. Co-inertia analyses suggested that bacterial-fungal variance was driven primarily by 12 bacterial operational taxonomic units (OTUs). Brome increased bacterial diversity via smooth brome litter in the A horizon and roots in the B horizon, which then reduced fungal diversity. Archaea increased abundance of several bacterial OTUs, and the key bacterial OTUs mediated changes in the fungi's response to invasion. Overall, native root diversity loss and bacterial mediation were more important drivers of fungal composition than were the direct effects of increases in smooth brome. Critically, native plant species displacement and root loss appeared to be the most important driver of fungal composition during invasion. This causal web likely gives rise to the plant-fungi feedbacks, which are an essential factor determining plant diversity in invaded grassland ecosystems.

Native fungal endophytes suppress an exotic dominant and increase plant diversity over small and large spatial scales.

Understanding community dynamics and processes, such as the factors that generate and maintain biodiversity, drive succession, and affect invasion susceptibility, is a central goal in ecology and evolution. While most studies of how species interactions affect communities have focused on highly visible macroorganisms, we show that mutualistic microfungal endophytes have community-level effects across their host plant's range and provide the first example of fungal endophytes enhancing plant diversity. A three-year field study in which we experimentally manipulated endophyte abundance in a native Californian grass showed that despite their minute biomass, endophytes dramatically increased plant community diversity (~110% greater increase with endophytes) by suppressing a dominant invasive grass, Bromus diandrus. This effect was also detectable, but smaller, across five additional common gardens spanning ecologically diverse habitats, different climates, and > 400 km of the host grass' range as well as at microspatial scales within gardens. Our study illustrates that mutualistic microbes, while often hidden players, can have unexpectedly large ecological impacts across a wide range of habitats and scales and may be important for promoting diverse communities and ecosystems.

Postdispersal Infection and Disease Development of Pyrenophora semeniperda in Bromus tectorum Seeds.

The Ascomycete fungus, Pyrenophora semeniperda, attacks a broad range of cool-season grasses. While leaf and predispersal infection of seeds (i.e., florets containing caryopses) have been previously characterized, little is known about the pathogenesis of mature seeds following dispersal. In this study, we examined infection and disease development of P. semeniperda on dormant seeds of Bromus tectorum. Inoculated seeds were hydrated at 20°C for up to 28 days. Disease development was characterized using scanning electron and light microscopy. P. semeniperda conidia germinated on the seed surface within 5 to 8 h. Hyphae grew on the seed surface and produced extracellular mucilage that eventually covered the seed. Appressoria formed on the ends of hyphae and penetrated through the lemma and palea, stomatal openings, and broken trichomes. The fungus then catabolized the endosperm, resulting in a visible cavity by 8 days. Pathogenesis of the embryo was associated with progressive loss of cell integrity and proliferation of mycelium. Beginning at approximately day 11, one to several stromata (approximately 150 µm in diameter and up to 4 mm in length) emerged through the lemma and palea. Degradation of embryo tissue was completed near 14 days. Conidiophores produced conidia between 21 and 28 days and often exhibited "Y-shaped" branching. This characterization of disease development corrects previous reports which concluded that P. semeniperda is only a weak seed pathogen with infection limited to the outermost seed tissues. In addition, the time required for disease development explains why infected dormant or slow-germinating seeds are most likely to experience mortality.

Relative abundance of and composition within fungal orders differ between cheatgrass (Bromus tectorum) and sagebrush (Artemisia tridentata)-associated soils.

Nonnative Bromus tectorum (cheatgrass) is decimating sagebrush steppe, one of the largest ecosystems in the Western United States, and is causing regional-scale shifts in the predominant plant-fungal interactions. Sagebrush, a native perennial, hosts arbuscular mycorrhizal fungi (AMF), whereas cheatgrass, a winter annual, is a relatively poor host of AMF. This shift is likely intertwined with decreased carbon (C)-sequestration in cheatgrass-invaded soils and alterations in overall soil fungal community composition and structure, but the latter remain unresolved. We examined soil fungal communities using high throughput amplicon sequencing (ribosomal large subunit gene) in the 0-4 cm and 4-8 cm depth intervals of six cores from cheatgrass- and six cores from sagebrush-dominated soils. Sagebrush core surfaces (0-4 cm) contained higher nitrogen and total C than cheatgrass core surfaces; these differences mirrored the presence of glomalin related soil proteins (GRSP), which has been associated with AMF activity and increased C-sequestration. Fungal richness was not significantly affected by vegetation type, depth or an interaction of the two factors. However, the relative abundance of seven taxonomic orders was significantly affected by vegetation type or the interaction between vegetation type and depth. Teloschistales, Spizellomycetales, Pezizales and Cantharellales were more abundant in sagebrush libraries and contain mycorrhizal, lichenized and basal lineages of fungi. Only two orders (Coniochaetales and Sordariales), which contain numerous economically important pathogens and opportunistic saprotrophs, were more abundant in cheatgrass libraries. Pleosporales, Agaricales, Helotiales and Hypocreales were most abundant across all libraries, but the number of genera detected within these orders was as much as 29 times lower in cheatgrass relative to sagebrush libraries. These compositional differences between fungal communities associated with cheatgrass- and sagebrush-dominated soils warrant future research to examine soil fungal community composition across more sites and time points as well as in association with native grass species that also occupy cheatgrass-invaded ecosystems.

Altered snowfall and soil disturbance influence the early life stage transitions and recruitment of a native and invasive grass in a cold desert.

Climate change effects on plants are expected to be primarily mediated through early life stage transitions. Snowfall variability, in particular, may have profound impacts on seedling recruitment, structuring plant populations and communities, especially in mid-latitude systems. These water-limited and frequently invaded environments experience tremendous variation in snowfall, and species in these systems must contend with harsh winter conditions and frequent disturbance. In this study, we examined the mechanisms driving the effects of snowpack depth and soil disturbance on the germination, emergence, and establishment of the native Pseudoroegnaria spicata and the invasive Bromus tectorum, two grass species that are widely distributed across the cold deserts of North America. The absence of snow in winter exposed seeds to an increased frequency and intensity of freeze-thaw cycles and greater fungal pathogen infection. A shallower snowpack promoted the formation of a frozen surface crust, reducing the emergence of both species (more so for P. spicata). Conversely, a deeper snowpack recharged the soil and improved seedling establishment of both species by creating higher and more stable levels of soil moisture availability following spring thaw. Across several snow treatments, experimental disturbance served to decrease the cumulative survival of both species. Furthermore, we observed that, regardless of snowpack treatment, most seed mortality (70-80%) occurred between seed germination and seedling emergence (November-March), suggesting that other wintertime factors or just winter conditions in general limited survival. Our results suggest that snowpack variation and legacy effects of the snowpack influence emergence and establishment but might not facilitate invasion of cold deserts.

Identification of the Infection Route of a Fusarium Seed Pathogen into Nondormant Bromus tectorum Seeds.

The genus Fusarium has a wide host range and causes many different forms of plant disease. These include seed rot and seedling blight diseases of cultivated plants. The diseases caused by Fusarium on wild plants are less well-known. In this study, we examined disease development caused by Fusarium sp. n on nondormant seeds of the important rangeland weed Bromus tectorum as part of broader studies of the phenomenon of stand failure or "die-off" in this annual grass. We previously isolated an undescribed species in the F. tricinctum species complex from die-off soils and showed that it is pathogenic on seeds. It can cause high mortality of nondormant B. tectorum seeds, especially under conditions of water stress, but rarely attacks dormant seeds. In this study, we used scanning electron microscopy (SEM) to investigate the mode of attack used by this pathogen. Nondormant B. tectorum seeds (i.e., florets containing caryopses) were inoculated with isolate Skull C1 macroconidia. Seeds were then exposed to water stress conditions (-1.5 MPa) for 7 days and then transferred to free water. Time lapse SEM photographs of healthy versus infected seeds revealed that hyphae under water stress conditions grew toward and culminated their attack at the abscission layer of the floret attachment scar. A prominent infection cushion, apparent macroscopically as a white tuft of mycelium at the radicle end of the seed, developed within 48 h after inoculation. Seeds that lacked an infection cushion completed germination upon transfer to free water, whereas seeds with an infection cushion were almost always killed. In addition, hyphae on seeds that did not initiate germination lacked directional growth and did not develop the infection cushion. This strongly suggests that the fungal attack is triggered by seed exudates released through the floret attachment scar at the initiation of germination. Images of cross sections of infected seeds showed that the fungal hyphae first penetrated the caryposis wall, then entered the embryo, and later ramified throughout the endosperm, completely destroying the seed.

Interspecific hybridization and bioactive alkaloid variation increases diversity in endophytic Epichloë species of Bromus laevipes.

Studying geographic variation of microbial mutualists, especially variation in traits related to benefits they provide their host, is critical for understanding how these associations impact key ecological processes. In this study, we investigate the phylogenetic population structure of Epichloë species within Bromus laevipes, a native cool-season bunchgrass found predominantly in California. Phylogenetic classification supported inference of three distinct Epichloë taxa, of which one was nonhybrid and two were interspecific hybrids. Inheritance of mating-type idiomorphs revealed that at least one of the hybrid species arose from independent hybridization events. We further investigated the geographic variation of endophyte-encoded alkaloid genes, which is often associated with key benefits of natural enemy protection for the host. Marker diversity at the ergot alkaloid, loline, indole-diterpene, and peramine loci revealed four alkaloid genotypes across the three identified Epichloë species. Predicted chemotypes were tested using endophyte-infected plant material that represented each endophyte genotype, and 11 of the 13 predicted alkaloids were confirmed. This multifaceted approach combining phylogenetic, genotypic, and chemotypic analyses allowed us to reconstruct the diverse evolutionary histories of Epichloë species present within B. laevipes and highlight the complex and dynamic processes underlying these grass-endophyte symbioses.

De novo genome assembly of the fungal plant pathogen Pyrenophora semeniperda.

Pyrenophora semeniperda (anamorph Drechslera campulata) is a necrotrophic fungal seed pathogen that has a wide host range within the Poaceae. One of its hosts is cheatgrass (Bromus tectorum), a species exotic to the United States that has invaded natural ecosystems of the Intermountain West. As a natural pathogen of cheatgrass, P. semeniperda has potential as a biocontrol agent due to its effectiveness at killing seeds within the seed bank; however, few genetic resources exist for the fungus. Here, the genome of P. semeniperda isolate assembled from sequence reads of 454 pyrosequencing is presented. The total assembly is 32.5 Mb and includes 11,453 gene models encoding putative proteins larger than 24 amino acids. The models represent a variety of putative genes that are involved in pathogenic pathways typically found in necrotrophic fungi. In addition, extensive rearrangements, including inter- and intrachromosomal rearrangements, were found when the P. semeniperda genome was compared to P. tritici-repentis, a related fungal species.

Inclusion of glycerol in forage diets increases methane production in a rumen simulation technique system.

We hypothesised that the inclusion of glycerol in the forage diets of ruminants would increase the proportion of propionate produced and thereby decrease in vitro CH4 production. This hypothesis was examined in the present study using a semi-continuous fermentation system (rumen simulation technique) fed a brome hay (8·5 g) and maize silage (1·5 g) diet with increasing concentrations (0, 50, 100 and 150 g/kg DM) of glycerol substituted for maize silage. Glycerol linearly increased total volatile fatty acids production (P<0·001). Acetate production was quadratically affected (P=0·023) and propionate and butyrate production was linearly increased (P<0·001). Glycerol linearly increased (P=0·011) DM disappearance from hay and silage. Crude protein disappearance from hay was not affected (P=0·789), but that from silage was linearly increased (P<0·001) with increasing glycerol concentrations. Neutral-detergent fibre (P=0·040) and acid-detergent fibre (P=0·031) disappearance from hay and silage was linearly increased by glycerol. Total gas production tended to increase linearly (P=0·061) and CH4 concentration in gas was linearly increased (P<0·001) by glycerol, resulting in a linear increase (P<0·001) in mg CH4/g DM digested. Our hypothesis was rejected as increasing concentrations of glycerol in a forage diet linearly increased CH4 production in semi-continuous fermenters, despite the increases in the concentrations of propionate. In conclusion, this apparent discrepancy is due to the more reduced state of glycerol when compared with carbohydrates, which implies that there is no net incorporation of electrons when glycerol is metabolised to propionate.

Diurnal patterns of productivity of arbuscular mycorrhizal fungi revealed with the Soil Ecosystem Observatory.

Arbuscular mycorrhizal (AM) fungi are the most abundant plant symbiont and a major pathway of carbon sequestration in soils. However, their basic biology, including their activity throughout a 24-h day : night cycle, remains unknown. We employed the in situ Soil Ecosystem Observatory to quantify the rates of diurnal growth, dieback and net productivity of extra-radical AM fungi. AM fungal hyphae showed significantly different rates of growth and dieback over a period of 24 h and paralleled the circadian-driven photosynthetic oscillations observed in plants. The greatest rates (and incidences) of growth and dieback occurred between noon and 18:00 h. Growth and dieback events often occurred simultaneously and were tightly coupled with soil temperature and moisture, suggesting a rapid acclimation of the external phase of AM fungi to the immediate environment. Changes in the environmental conditions and variability of the mycorrhizosphere may alter the diurnal patterns of productivity of AM fungi, thereby modifying soil carbon sequestration, nutrient cycling and host plant success.

Consequences of pathogen spillover for cheatgrass-invaded grasslands: coexistence, competitive exclusion, or priority effects.

With the rise in species invasions and emerging infectious diseases, pathogen spillover from abundant reservoir hosts to their competitors is increasingly common. Although the potential for pathogen spillover is widespread, its consequences for host community composition remain poorly understood. To address this gap, I examine the consequences of fungal seed pathogen spillover from an exotic annual grass (cheatgrass) to a native perennial bunchgrass in the Intermountain West, United States, using a model. Integrating generalist pathogens with broader coexistence theory, the model measures the pathogen's effect on host niche differences and fitness differences, which determine the outcome of competition. The model demonstrates that the consequences of pathogen spillover depend on host differences in species-specific transmission and disease tolerance. Counterintuitively, spillover can lead to coexistence, native grass exclusion, or priority effects, in which either species can exclude the other when initially more dominant. Cheatgrass has higher tolerance for infection, which could lead to competitive dominance or to coexistence if the native grass has a fecundity or survival advantage. In sum, multihost pathogens can affect host communities in a range of ways, depending on the specific mechanism of spillover.

Comparative genomics of multiple strains of Pseudomonas cannabina pv. alisalensis, a potential model pathogen of both monocots and dicots.

Comparative genomics of closely related pathogens that differ in host range can provide insights into mechanisms of host-pathogen interactions and host adaptation. Furthermore, sequencing of multiple strains with the same host range reveals information concerning pathogen diversity and the molecular basis of virulence. Here we present a comparative analysis of draft genome sequences for four strains of Pseudomonas cannabina pathovar alisalensis (Pcal), which is pathogenic on a range of monocotyledonous and dicotyledonous plants. These draft genome sequences provide a foundation for understanding host range evolution across the monocot-dicot divide. Like other phytopathogenic pseudomonads, Pcal strains harboured a hrp/hrc gene cluster that codes for a type III secretion system. Phylogenetic analysis based on the hrp/hrc cluster genes/proteins, suggests localized recombination and functional divergence within the hrp/hrc cluster. Despite significant conservation of overall genetic content across Pcal genomes, comparison of type III effector repertoires reinforced previous molecular data suggesting the existence of two distinct lineages within this pathovar. Furthermore, all Pcal strains analyzed harbored two distinct genomic islands predicted to code for type VI secretion systems (T6SSs). While one of these systems was orthologous to known P. syringae T6SSs, the other more closely resembled a T6SS found within P. aeruginosa. In summary, our study provides a foundation to unravel Pcal adaptation to both monocot and dicot hosts and provides genetic insights into the mechanisms underlying pathogenicity.

Elevated CO2 spurs reciprocal positive effects between a plant virus and an arbuscular mycorrhizal fungus.

Plants form ubiquitous associations with diverse microbes. These interactions range from parasitism to mutualism, depending partly on resource supplies that are being altered by global change. While many studies have considered the separate effects of pathogens and mutualists on their hosts, few studies have investigated interactions among microbial mutualists and pathogens in the context of global change. Using two wild grass species as model hosts, we grew individual plants under ambient or elevated CO(2), and ambient or increased soil phosphorus (P) supply. Additionally, individuals were grown with or without arbuscular mycorrhizal inoculum, and after 2 wk, plants were inoculated or mock-inoculated with a phloem-restricted virus. Under elevated CO(2), mycorrhizal association increased the titer of virus infections, and virus infection reciprocally increased the colonization of roots by mycorrhizal fungi. Additionally, virus infection decreased plant allocation to root biomass, increased leaf P, and modulated effects of CO(2) and P addition on mycorrhizal root colonization. These results indicate that plant mutualists and pathogens can alter each other's success, and predict that these interactions will respond to increased resource availability and elevated CO(2). Together, our findings highlight the importance of interactions among multiple microorganisms for plant performance under global change.

Resource availability and imbalance affect plant-mycorrhizal interactions: a field test of three hypotheses.

Ecological stoichiometry can explain major trends in how interactions among species change across fertility gradients, but important questions remain. For example, stoichiometry predicts that fertilization should cause plants to reduce carbon allocation to arbuscular mycorrhizal fungi and, consequently, reduce fungal abundance, but responses in the field are highly variable. In a field experiment, we tested three hypotheses that could drive this variation: (1) fungi are nitrogen limited in very nitrogen-poor soils, so fertilization increases their abundance; (2) the N:P ratio of fertilization affects plant carbon allocation to fungi; (3) plant species differences affect fungal response. Our results support all three hypotheses: stoichiometry and species idiosyncrasies jointly determined fungal response to fertilization. We provide field evidence in support of the hypothesis that nitrogen can limit fungal abundance in temperate grasslands. We also show that fungal abundance in soil (hyphal length) differed beneath two dominant plant species: big bluestem (Andropogon gerardii) and smooth brome (Bromus inermis). These grass species also differed in the degree to which they reduced root colonization with fertilization, but these differences in allocation did not lead to differential responses to fertilization in terms of fungal abundance in the soil. This study shows that, while ecological stoichiometry is a useful framework for understanding the effects of eutrophication on this important and widespread species interaction, including these subtleties can increase the predictive power of the theory.

Arbuscular mycorrhizal fungal community differs between a coexisting native shrub and introduced annual grass.

Arbuscular mycorrhizal fungi (AMF) have been implicated in non-native plant invasion success and persistence. However, few studies have identified the AMF species associating directly with plant invaders, or how these associations differ from those of native plant species. Identifying changes to the AMF community due to plant invasion could yield key plant-AMF interactions necessary for the restoration of native plant communities. This research compared AMF associating with coexisting Bromus tectorum, an invasive annual grass, and Artemisia tridentata, the dominant native shrub in western North America. At three sites, soil and root samples from Bromus and Artemisia were collected. Sporulation was induced using trap cultures, and spores were identified using morphological characteristics. DNA was extracted from root and soil subsamples and amplified. Sequences obtained were aligned and analyzed to compare diversity, composition, and phylogenetic distance between hosts and sites. Richness of AMF species associated with Artemisia in cultures was higher than AMF species associated with Bromus. Gamma diversity was similar and beta diversity was higher in AMF associated with Bromus compared to Artemisia. AMF community composition differed between hosts in both cultures and roots. Two AMF species (Archaeospora trappei and Viscospora viscosum) associated more frequently with Artemisia than Bromus across multiple sites. AMF communities in Bromus roots were more phylogenetically dispersed than in Artemisia roots, indicating a greater competition for resources within the invasive grass. Bromus associated with an AMF community that differed from Artemisia in a number of ways, and these changes could restrict native plant establishment.

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.

Population genetic structure of the seed pathogen Pyrenophora semeniperda on Bromus tectorum in western North America.

We examined genetic variation in the ascomycete pathogen Pyrenophora semeniperda cultured from seeds of the invasive grass Bromus tectorum in the Intermountain West of North America. We sequenced the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA genome in 417 monoconidial cultures collected from 20 sites in Washington, Idaho, Utah and Colorado, USA. ITS sequence diversity was surprisingly high; 12 unique haplotypes were identified, averaging 1.3% pairwise sequence divergence. All sites had at least two haplotypes present, and three sites had seven or more. One haplotype composed 60% of the isolates and occurred at all 20 locations; the remaining haplotypes generally occurred at low frequencies within sites but at multiple sites throughout the region. Sites in Washington and Idaho were more diverse than those in Utah and Colorado, averaging two more haplotypes and 67% more pairwise differences among haplotypes at a site. Analysis of molecular variance (AMOVA) indicated that more than 80% of the genetic variation was found within sampling locations, while 7-11% of the variation can be attributed to differences between northern (Washington and Idaho) and southern (Utah and Colorado) populations. The wide distribution of even uncommon haplotypes among sampling sites and weak correlations between genetic and geographic distances among populations (< 0.2) suggested that these populations recently were established from a common source. We hypothesize that the strains of P. semeniperda infecting B. tectorum in western North America probably arrived with the invasive grass from its native Eurasian range.