Pyrenophora semeniperda is a generalist fungal pathogen that occurs primarily on monocot seed hosts. It is in the phylum Ascomycota, which includes both self-compatible (homothallic) and self-incompatible (heterothallic) species. Homothallic fungal species contain complementary mating-type (MAT) idiomorphs in a single unikaryotic strain, while heterothallic strains contain a single MAT idiomorph requiring interaction between strains of complementary mating-types for sexual reproduction to occur. Because the majority of P. semeniperda strains contained either MAT1 or MAT2, this species was provisionally categorized as heterothallic. However, many strains contain both MAT idiomorphs and appear to be homothallic. These results warranted a closer look at the MAT idiomorphs and the structure of the P. semeniperda genome in order to assure accurate characterization of the MAT locus. Additionally, an assessment of the geographic distribution of MAT idiomorphs provides us with insight into the genetic diversity of P. semeniperda and the reproductive strategies that it employs. In this study, we characterized the P. semeniperda MAT locus and assessed the idiomorph distribution of 514 isolates from 25 P. semeniperda populations collected from infected Bromus tectorum (cheatgrass) seeds. Additionally, we used simple sequence repeat (SSR) and MAT idiomorph length polymorphisms to demonstrate the existence of dikaryotic strains and pseudohomothallism in this fungus. We identified a unique variable number tandem repeat (VNTR) within each idiomorph of the MAT locus of P. semeniperda. Presence of the VNTR in all MAT loci analyzed from strains collected in the Intermountain West suggests ancient proliferation of this repeat. The persistence and effectiveness of P. semeniperda strains in the cheatgrass pathosystem depend not only on the density of the fungus in the soil, but also on the genetic heterogeneity of each population. Our study suggests that P. semeniperda genetic diversity is increased both through MAT locus-dependent sexual reproduction and asexually through anastomosis.

Contributing to the success of Bromus tectorum in the Intermountain West may be a mechanism called apparent competition, which occurs when one species increases the pressure of a consumer on a second species. This indirect interaction has been documented only a few times in invasive plant systems, and never in a fungal pathosystem. We examined the effects of the invasive annual Bromus tectorum and predation by the seed pathogen Pyrenophora semeniperda on seedling emergence and survival for two native grasses (Pseudoroegneria spicata and Elymus elymoides), by manipulating B. tectorum densities and P. semeniperda inoculum loads in randomized plots. Identical field studies were conducted in Skull Valley, Utah (xeric site) and Sprague, Washington (mesic site). The addition of inoculum decreased emergence of native grass seedlings at both sites and increased the amount of unemerged native seeds that were killed by P. semeniperda. Higher densities of B. tectorum decreased native grass survival at the mesic site and increased survival at the xeric site probably due to the beneficial effects of B. tectorum litter on soil moisture. At both sites, there were more B. tectorum seeds found in the seed banks in plots with high B. tectorum densities than in low-density plots. This indicates an increase in available prey for P. semeniperda. There was a much lower level of infection in B. tectorum seed bank seeds at the mesic site than at the xeric site. The high level of ungerminated native seeds killed by background levels of P. semeniperda, combined with the increase in available prey for the fungus in high-density B. tectorum plots, shows that apparent competition may play a role, along with direct competition, in the success of B. tectorum. This interaction is important to consider when dealing with control of B. tectorum.

Bromus tectorum and other annual brome grasses have invaded many ecosystems of the western United States, and because of an annual-grass influenced alteration of the natural fire cycle on arid western range lands near monocultures are created and conditions in which the native vegetation cannot compete are established. Each year thousands of hectares become near monocultures of annual brome grasses. Pyrenophora semeniperda, a generalist seed pathogen of annual grasses, shows major potential as a possible mycoherbicide that could help in reducing the monocultures created by annual grasses. The purpose of this research was to identify the requirements for isolating cultures of P. semeniperda, search for a hypervirulent strain, and evaluate its effect in the field. The techniques for isolating the fungus have evolved and become more efficient. The first two years of working with P. semeniperda resulted in 11 isolates. During the third year of this study, we developed a single spore isolation technique that resulted in 480 additional isolates. Virulence screening resulted in detection of a range of isolate ability to kill non-dormant B. tectorum seeds. Ninety-two isolates represented a range of virulence from 0-44%. The variation in virulence was expressed mostly within populations rather than between populations. Similarly, virulence varied significantly within Internal Transcribed Spacer (ITS) genotypes and habitats but not between them. When conidial inoculum was applied in the field there was no observed difference in disease incidence between different levels of inoculum. This is thought to have been due to applying the inoculum under conditions in which most in situ seeds were infected and killed by already high field inoculum loads. While additional field trials are needed to optimize the inoculum effectiveness, the overall results of this research provide a good foundation for using P. semeniperda as a biological control for seed banks of annual brome grasses.

Variable mortality of Pyrenophora semeniperda--infected Bromus tectorum seeds has been referred to as a "race for survival", stating that seeds that germinate quickly are more likely to escape pathogen-caused mortality. Dormancy status is not the only variable determining outcomes within the Bromus-Pyrenophora pathosystem. Varying temperature and exposure to water may strongly influence germination outcomes of B. tectorum when in the presence of P. semeniperda. Low water potentials characteristic of semi-arid soils are often over-looked in the context of seed pathogens, and are ecologically relevant- especially for plant species that inhabit intermittently dry environments. To adequately characterize the Bromus tectorum-Pyrenophora semeniperda pathosystem, four studies were conducted to address the following questions: (1) do temperature, water potential, and dormancy status influence germination outcomes in the Bromus-Pyrenophora pathosystem, (2) do repeated wetting-drying scenarios influence germination outcomes of infected B. tectorum seeds following dehydration at low water potentials similar to those found in the field (i.e., -4 through -150 MPa), (3) can we accurately characterize the asexual life cycle of P. semeniperda on a dormant B. tectorum seed, determining when infection takes place, and what occurs during disease development in continuously hydrated conditions, and (4) how does disease development of P. semeniperda influence the B. tectorum seed embryo and endosperm. All studies were conducted using dormant and/or non-dormant B. tectorum seeds and an intermediate strain of P. semeniperda. Study one used varying temperatures (5-20°C), and five water potentials (0, -0.5, -1, -1.5, -2 MPa) (achieved using PEG 8000). Inoculated seeds were exposed to low water potentials at various temperatures for 7, 14, 21, or 28 days then re-hydrated for 28 days. In the second study, seeds were incubated at 20°C at four nominal water potentials (-4, -10, -40, or -150 MPa) following 8 or 24 hours of initial hydration. Seeds were dehydrated for 1, 7, 14, or 21 days, then re-hydrated. In study three, inoculated seeds were chemically fixed between days 0 and 21 and viewed with a scanning electron microscope. In the fourth study, infected seeds were frozen with liquid nitrogen following 3, 8, and 14 days of disease development, then cross sectioned longitudinally and laterally prior to chemical fixation. Results indicate that non-dormant seeds escape death by germinating rapidly under favorable conditions, that incubation at low water potentials greatly increases seed mortality, that -10 MPa is near the threshold for full pathogen activity, and at water potentials lower than -40 MPa, P. semeniperda may successfully survive severe dehydration if previous hydration resulting in infection has occurred. SEM images indicate that mycelia penetration occurs within 8-24 hours, and that mycelium may penetrate all opening in the seed (i.e., stomata, cracks). Development of P. semeniperda is shown to cause significant damage to the endosperm and embryo within 8 days. As starch is consumed, the endosperm collapses leaving a hollow middle. The embryo is more resilient, but gradually deforms and deteriorates.

Variable mortality of Pyrenophora semeniperda–infected Bromus tectorum seeds has been referred to as a “race for survival.” Dormant seeds are highly susceptible to P. semeniperda infection. While much is known about primary dormancy little is known about secondary dormancy in B. tectorum seeds. Dormancy status is not the only variable determining outcomes within the Bromus - Pyrenophora pathosystem. Varying temperature and intermittent hydration may strongly influence germination outcomes of B. tectorum in the presence of P. semeniperda. While it has long been assumed that B. tectorum seeds are infected by P. semeniperda in the fall it was recently suggested that seeds may be infected in the summer; however, there is little evidence to support this. To further characterize the Pyrenophora semeniperda - Bromus tectorum pathosystem two studies were conducted to address the following: (1) characterization of secondary dormancy in B. tectorum seeds and (2) summer interactions between host and pathogen after summer inoculation. Studies were conducted using dormant and/or non-dormant B. tectorum (along with B. rubens in one study) seeds and two strains of P. semeniperda. Study one used laboratory and field experiments to characterize secondary dormancy in B. tectorum seeds in terms of temperature (0.5-20°C), and water potential (-2.0-0 MPa). Data was used in repeated probit regression analysis to determine hydrothermal parameters (ψb(50), σψb, θHT) for secondary dormancy induction and loss. In the second study seeds were inoculated with one of two strains of P. semeniperda then exposed to intermittent hydration or dry storage at warm temperatures (30-60°C). After treatment seeds were rehydrated and outcomes observed. Optimum conditions for secondary dormancy induction were incubation at -1.0 MPa at 5°C. Seeds were likely to enter secondary dormancy through the cold winter months indicated by an increase or more positive ψb(50), while a decrease or more negative ψb(50) is associated with dormancy loss which is generally observed in the hot, dry summer months. When seeds were inoculated in the summer they only escaped death when summer conditions were ideal for after-ripening which allowed them to germinate rapidly under favorable autumn conditions. However, the pathogen caused high seed mortality no matter the treatment when disease progression advanced enough to inhibit seed germination. Thus this research shows that in areas with frequent summer rain storms, it would be highly advantageous to apply P. semeniperda as a biocontrol on seeds at maturity.