Pyrenophora Semeniperda Research Articles

In a survey of barley leaf spot diseases across East Azerbaijan province in Iran, barley plants with brown leaf spot, were collected from barley fields in the Spiran region, the central district of Tabriz County, East Azerbaijan Province, Iran. The disease incidence was 30 percent in sampled areas. Fungal colonies with similar growth pattern were isolated from symptomatic leaves. The isolates were characterized based on a combination of morphological data and sequence data of ITS-rDNA region as P. semeniperda. Pathogenicity assay was fulfilled using the seed infection method under greenhouse conditions. Correspondingly, P. semeniperda stromata were developed on 90% of intact and 95% of scarifies barley seeds and they failed to germinate. Koch’s postulates were fulfilled by re-isolation of the causal agent from inoculated seeds. To the best of our knowledge, P. semeniperda represents a new pathogen on barley in Iran. However, geographical distribution, host range and economic impact of this pathogen on major barley producing areas in Iran remain to be studied.

Pyrenophoric acid (P-Acid), P-Acid B, and P-Acid C are three phytotoxic sesquiterpenoids produced by the ascomycete seed pathogen Pyrenophora semeniperda, a fungus proposed as a mycoherbicide for biocontrol of cheatgrass, an extremely invasive weed. When tested in cheatgrass bioassays, these metabolites were able to delay seed germination, with P-Acid B being the most active compound. Here, we have investigated the cross-kingdom activity of P-Acid B and its mode of action, and found that it activates the abscisic acid (ABA) signaling pathway in order to inhibit seedling establishment. P-Acid B inhibits seedling establishment in wild-type Arabidopsis thaliana, while several mutants affected in the early perception as well as in downstream ABA signaling components were insensitive to the fungal compound. However, in spite of structural similarities between ABA and P-Acid B, the latter is not able to activate the PYR/PYL family of ABA receptors. Instead, we have found that P-Acid B uses the ABA biosynthesis pathway at the level of alcohol dehydrogenase ABA2 to reduce seedling establishment. We propose that the fungus P. semeniperda manipulates plant ABA biosynthesis as a strategy to reduce seed germination, increasing its ability to cause seed mortality and thereby increase its fitness through higher reproductive success.

Abstract Downy brome (Bromus tectorum L., syn. cheatgrass) is a winter annual grass that invades North American cropping, forage, and rangeland systems. Control is often difficult to achieve, because B. tectorum has a large seedbank, which results in continuous propagule pressure. Pyrenophora semeniperda (Brittlebank and Adam) Shoemaker, a soilborne fungal pathogen, has been investigated as a biological control for B. tectorum, because it can kill seeds that remain in the seedbank, thereby reducing propagule pressure. Temperature influences P. semeniperda and has not been investigated in the context of seeds collected from different B. tectorum locations, that may vary in susceptibility to infection. We compared the effects of temperature (13, 17, 21, 25 C) and B. tectorum seed locations (range, crop, subalpine) with different mean seed weights on infection rates of P. semeniperda using a temperature-gradient table. Infection differed by seed location (P < 0.001) and temperature (P < 0.001), with lighter-weight seeds (i.e., range and subalpine) more susceptible to P. semeniperda infection. Infection increased as temperature increased and was higher at 21 C (66.7 ± 6.7%) and 25 C (73.3 ± 6.0%). Germination was affected by seed location (P < 0.001) and temperature (P = 0.019). Germination was highest for the crop seed location (45.4 ± 4.2%) and overall decreased at higher temperatures (21 and 25 C). Our results suggest that B. tectorum seeds from a crop location are less affected by P. semeniperda than those from range and subalpine locations. Moreover, this demonstrates a temperature-dependent effect on all populations.

Mating strategies contributing to a balance between inbreeding and outcrossing are understudied in all but a few model fungal pathogens. This study examined factors that influence the occurrence of the sexual state of Pyrenophora semeniperda. It was consistently found to have functional copies of the MAT1‐1 and MAT1‐2 idiomorphs essential for sexual reproduction, despite considerable polymorphism in both single nucleotide polymorphisms (SNPs) and number of 18‐base minisatellite repeats. The two idiomorphs occurred at approximately equal frequencies across 25 populations on Bromus tectorum seeds in the western United States, suggesting maintenance of sexual reproduction. The putative mating system is described as facultative pseudohomothallism, with only one of the two MAT1 idiomorphs found in a nucleus. Unikaryotic strains of opposite mating type can potentially mate, as can nuclei of opposite mating type in a thallus that results from anastomosis between vegetatively compatible unikaryotic strains. Strains shown to be dikaryotic using simple sequence repeat (SSR) markers may contain either or both MAT1 idiomorphs. Most populations consist of a mixture of MAT1‐1, MAT1‐2 and MAT1‐1/MAT1‐2 genotypes. A possible constraint on recombination is the presence of multiple strain groups characterized by ITS haplotype. These are apparently vegetatively incompatible, as even dikaryotic strains are invariably composed of a single ITS haplotype. Different ITS haplotypes also have unique combinations of MAT1‐1 and MAT1‐2 allelic variants, suggesting that perhaps these strain groups are also sexually incompatible. Phylogenetic analysis using both genome‐wide SNP/indel polymorphisms and SSR markers demonstrated genetic divergence among ITS haplotypes, supporting the hypothesis that these strain groups may represent cryptic species.

A proposed mechanism for high pathogen-caused mortality in the seed bank of an invasive annual grass

Abstract Niche theory predicts that when two species exhibit major niche overlap, one will eventually be eliminated through competitive exclusion. Thus, some degree of niche specialization is required to facilitate coexistence. We examined whether two important seed bank pathogens on the invasive winter annual grass Bromus tectorum (cheatgrass, downy brome) exhibit niche specialization. These pathogens utilize seed resources in complementary ways. Pyrenophora semeniperda is specialized to attack dormant seeds. It penetrates directly through the seed coverings. Hyphae ramify first through the endosperm and then throughout the seed. Seed death results as the embryo is consumed. In contrast, the Fusarium seed rot pathogen (Fusarium sp.) is specialized to attack non-dormant seeds in the early stages of germination. It cannot penetrate seed coverings directly. Instead, it responds to a cue emanating from the radicle end with directional hyphal growth and subsequent penetration at the point of radicle emergence, causing seed death. Non-dormant seeds usually escape P. semeniperda through germination even if infected because it develops more slowly than Fusarium. When water stress slows non-dormant seed germination, both P. semeniperda and Fusarium can attack and cause seed mortality more effectively. The Fusarium seed rot pathogen can sometimes reach epidemic levels and may result in B. tectorum stand failure (‘die-off’). Stands usually re-establish from the persistent seed bank, but if P. semeniperda has also reached high levels and eliminated the seed bank, a die-off can persist indefinitely.

Generalist plant pathogens may have wide host ranges, but many exhibit varying degrees of host specialization, with multiple pathogen races that have narrower host ranges. These races are often genetically distinct, with each race causing highest disease incidence on its host of origin. We examined host specialization in the seed pathogen Pyrenophora semeniperda by reciprocally inoculating pathogen strains from Bromus tectorum and from four other winter annual grass weeds (Bromus diandrus, Bromus rubens, Bromus arvensis and Taeniatherum caput-medusae) onto dormant seeds of B. tectorum and each alternate host. We found that host species varied in resistance and pathogen strains varied in aggressiveness, but there was no evidence for host specialization. Most variation in aggressiveness was among strains within populations and was expressed similarly on both hosts, resulting in a positive correlation between strain-level disease incidence on B. tectorum and on the alternate host. In spite of this lack of host specialization, we detected weak but significant population genetic structure as a function of host species using two neutral marker systems that yielded similar results. This genetic structure is most likely due to founder effects, as the pathogen is known to be dispersed with host seeds. All host species were highly susceptible to their own pathogen races. Tolerance to infection (i.e., the ability to germinate even when infected and thereby avoid seed mortality) increased as a function of seed germination rate, which in turn increased as dormancy was lost. Pyrenophora semeniperda apparently does not require host specialization to fully exploit these winter annual grass species, which share many life history features that make them ideal hosts for this pathogen.

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.

Pyrenophora semeniperda, an important pathogen in Bromus tectorum seed banks in semi‐arid western North America, exhibits >4‐fold variation in mycelial growth rate. Host seeds exhibit seasonal changes in dormancy that affect the risk of pathogen‐caused mortality. The hypothesis tested is that contrasting seed dormancy phenotypes select for contrasting strategies for increasing pathogen fitness, and that increased fitness on nondormant seeds involves a resource trade‐off between toxin production and growth. The strategy for successfully attacking rapidly germinating nondormant seeds at high inoculum loads in autumn involves increased post‐infection aggressiveness to prevent seed escape through germination. An earlier study demonstrated that slow‐growing strains caused higher mortality than faster‐growing strains on nondormant host seeds at high inoculum loads. In this study, production of the toxin cytochalasin B was significantly higher in slower‐growing strains, and was induced only in seeds or in seed‐constituent‐containing media. Its production was reduced in vivo by Bromus tectorum seeds, suggesting direct involvement in pathogenesis on seeds. Fast‐growing strains caused significantly higher mortality than slow‐growing strains at low inoculum loads on dormant seeds, which apparently have resistance that is overcome at high loads or through rapid mycelial proliferation. In a co‐inoculation study, the fast‐growing isolate produced 3 × more stromata than the slow‐growing isolate on dormant seeds, whereas the slow‐growing isolate was twice as successful on nondormant seeds. These results provide evidence that mycelial growth rate variation and associated variation in cytochalasin B production represent a trade‐off maintained through temporally varying selection resulting from seasonal variation in host seed dormancy status.

Hydrothermal time models for conidial germination and mycelial growth of the seed pathogen Pyrenophora semeniperda

Two new phytotoxic sesquiterpenoid acids, named pyrenophoric acids B and C, were isolated together with the related pyrenophoric and abscisic acids from solid Bromus tectorum (cheatgrass) seed culture of the seed pathogen Pyrenophora semeniperda. This fungus has been proposed as a mycoherbicide for biocontrol of cheatgrass (Bromus tectorum), a Eurasian annual grass that has become invasive in rangelands and is also a serious agricultural weed in the western U.S. Pyrenophoric acids B and C were characterized by spectroscopic methods (NMR and HR ESIMS) as (2Z,4E)-5-[(1R*,4R*,6R*)-1,4-dihydroxy-2,2,6-trimethylcyclohexyl]-3-methylpenta-2,4-dienoic and (2Z,4E)-5-[(1S*,3S*,4R*,6S*)-3,4-dihydroxy-2,2,6-trimethylcyclohexyl]-3-methylpenta-2,4-dienoic acids, respectively. Cytochalasins A, B, F, and Z3, as well as deoxaphomin and pyrenophoric acid, all previously isolated from P. semeniperda grown on wheat seed, were also isolated from cheatgrass seed culture. In a cheatgrass seedling bioassay at 10(-3) M, pyrenophoric acid B showed higher coleoptile toxicity than pyrenophoric acid, while pyrenophoric acid C showed lower phytotoxicity. Abscisic acid was by far the most active compound.

SummaryBromus tectorum is a winter annual grass that affects rangeland in western North America. A glasshouse pot experiment was conducted that integrated imazapic application and inoculation of the soil‐borne fungal pathogen, Pyrenophora semeniperda, for the purpose of providing greater control of B. tectorum. We hypothesised that P. semeniperda inoculation would reduce B. tectorum emergence and integration of imazapic and P. semeniperda would result in a greater reduction in B. tectorum biomass and density compared with either treatment applied alone. This study revealed that P. semeniperda significantly reduced B. tectorum emergence and density and the responses were greatest for seed placed below the soil surface. Further, B. tectorum biomass was similar between imazapic and P. semeniperda treatments. This indicates that P. semeniperda could be applied in advance of B. tectorum germination and emergence. After emergence, imazapic application could reduce B. tectorum biomass and kill seedlings. A two‐pronged approach to controlling B. tectorum that combines P. semeniperda inoculation and post‐emergent imazapic application may provide a greater opportunity to limit invasion of this weed in rangeland of western North America. Future work should be directed towards the pathogen–plant relationship and how it relates to integrating biological control with traditional methods, towards the effect of varying P. semeniperda inoculum and imazapic rates and lastly, to how environmental conditions in the field may affect implementation and efficacy of this two‐pronged approach.

A new phytotoxic sesquiterpenoid penta-2,4-dienoic acid, named pyrenophoric acid, was isolated from solid wheat seed culture of Pyrenophora semeniperda, a fungal pathogen proposed as a mycoherbicide for biocontrol of cheatgrass (Bromus tectorum) and other annual bromes. These bromes are serious weeds in winter cereals and also on temperate semiarid rangelands. Pyrenophoric acid was characterized as (2Z,4E)-5-[(7S,9S,10R,12R)-3,4-dihydroxy-2,2,6-trimethylcyclohexyl)]-3-methylpenta-2,4-dienoic acid by spectroscopic and chemical methods. The relative stereochemistry of pyrenophoric acid was assigned using 1H,1H couplings and NOESY experiments, while its absolute configuration was determined by applying the advanced Mosher's method. Pyrenophoric acid is structurally quite closely related to the plant growth regulator abscisic acid. When bioassayed in a cheatgrass coleoptile elongation test at 10(-3) M, pyrenophoric acid showed strong phytotoxicity, reducing coleoptile elongation by 51% relative to the control. In a mixture at 10(-4) M, its negative effect on coleoptile elongation was additive with that of cytochalasin B, another phytotoxic compound found in the wheat seed culture extract of this fungus, demonstrating that the extract toxicity observed in earlier studies was due to the combined action of multiple phytotoxic compounds.

Generalist pathogens can exhibit differential success on different hosts, resulting in complex host range patterns. Several factors operate to reduce realized host range relative to potential host range, particularly under field conditions. We explored factors influencing host range of the naturally occurring generalist ascomycete grass seed pathogen Pyrenophora semeniperda .W e measured potential host range in laboratory experiments at high inoculum loads with 26 grass species, including the primary host Bromus tectorum, and developed models to predict susceptibility and tolerance based on host traits, including germination speed, seed hardness, seed size, and phylogenetic relations. We also examined pathogen and host density effects on infection and mortality. All species tested were at least somewhat susceptible to the pathogen at high inoculum loads, but both infection and mortality varied widely. Species more closely related to the original host (B. tectorum) were more susceptible to infection, whereas species with slower germination were less tolerant and therefore more likely to suffer mortality. Infection and mortality were sharply reduced as inoculum load was reduced. Intermediate loads had major negative impacts on dormant B. tectorum seeds but generally minimal effects on native species. In addition, field seed bank studies determined that P. semeniperda rarely exploits native grass species as hosts. This marked reduction in realized host range relative to potential host range indicates that laboratory host range studies are potentially a poor predictor of either the current or possible future realized host range for wildland plant pathogens.

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.

Spirostaphylotrichin W, a spirocyclic γ-lactam isolated from liquid culture of Pyrenophora semeniperda, a potential mycoherbicide for cheatgrass (Bromus tectorum) biocontrol

Invasive plants exhibit both direct and indirect negative effects on recruitment of natives following invasion. We examined indirect effects of the invader Bromus tectorum (cheatgrass) on seed fates of two native grass species, Elymus elymoides and Pseudoroegneria spicata, by removing B. tectorum and by adding inoculum of the shared seed pathogen Pyrenophora semeniperda in factorial experiments at xeric and mesic field sites. We also included a supplemental watering treatment to increase emergence and also the potential for pathogen escape. We recorded emergence and survival of native seedlings and also determined the fate of unemerged seeds. At the xeric site, Pyrenophora-caused mortality was high (34%), and effects of other pathogens and failed emergence of germinants were smaller. Cheatgrass removal negatively affected both emergence (35 vs. 25%) and spring survival (69 vs. 42%). Pyrenophora-caused seed mortality increased with inoculum augmentation for both species (22 vs. 47% overall), but emergence was negatively impacted only for P. spicata (20 vs. 34%). At the mesic site, Pyrenophora-caused mortality was low (6%). Cheatgrass removal doubled emergence (26 vs. 14%). Seed mortality increased significantly with inoculum augmentation for P. spicata (12 vs. 5%) but not E. elymoides, while emergence was not significantly affected in either species. A large fraction of seeds produced germinants that failed to emerge (37%), while another large fraction (35%) was killed by other pathogens. We conclude that facilitation by cheatgrass at the xeric site but interference at the mesic site was probably mediated through litter effects that could be ameliorative or suppressive. Apparent competition between cheatgrass and native grasses could occur through Pyrenophora, especially in a xeric environment, but effects were weak or absent at emergence. This was probably because Pyrenophora attacks the same slow-germinating fraction that is subject to pre-emergence mortality from other causes, including attack by other pathogens such as Fusarium.

How pathogen spillover influences host community diversity and composition is poorly understood. Spillover occurs when transmission from a reservoir host species drives infection in another host species. In cheatgrass-invaded grasslands in the western United States, a fungal seed pathogen, black fingers of death (Pyrenophora semeniperda), spills over from exotic cheatgrass (Bromus tectorum) to native perennial bunchgrasses such as squirreltail (Elymus elymoides). Previous theoretical work based on this system predicts that pathogens that spill over can favor either host coexistence, the exclusion of either host species, or priority effects, depending on species-specific transmission rates and pathogen tolerance. Here, these model predictions were tested by parameterizing a population growth model with field data from Skull Valley, Utah, USA. The model suggests that, across the observed range of demographic variation, the pathogen is most likely to provide a net benefit to squirreltail and a net cost to cheatgrass, though both effects are relatively weak. Although cheatgrass (the reservoir host) is more tolerant, squirreltail is far less susceptible to infection, and its long-lived adult stage buffers population growth against seed losses to the pathogen. This work shows that, despite pathogen spillover, the shared pathogen promotes native grass persistence by reducing exotic grass competition. Counterintuitively, the reservoir host does not necessarily benefit from the presence of the pathogen, and may even suffer greater costs than the nonreservoir host. Understanding the consequences of shared pathogens for host communities requires weighing species differences in susceptibility, transmission, and tolerance using quantitative models.

Pyrenophora semeniperda is abundant in soil seed banks of Bromus tectorum, where it kills a fraction of seeds throughout the year. The pathogen engages in a race with host seeds for endosperm resources; the pathogen success is negatively correlated with seed germination speed. We developed a deterministic model to predict pathosystem outcomes (seed death versus seed escape), using seed bank data from 80 sites collected over a 13-year period. The response variable (killed seeds in the spring seed bank) was regressed on multiple predictor variables (pathogen and host densities at seed dispersal, amount and timing of precipitation). Increased mortality was associated with high seed rain, high pathogen density, and low autumn precipitation. On xeric sites, a positive feedback loop between pathogen and host is created by a large carryover seed bank containing secondarily dormant seeds vulnerable to fungal attack and results in higher inoculum loads at seed dispersal the following year.

Bromus tectorum is a highly invasive annual grass. The fungal pathogen Pyrenophora semeniperda can kill a large fraction of B. tectorum seeds. Outcomes in this pathosystem are often determined by the speed of seed germination. In this paper we extend previous efforts to describe the pathosystem by characterising secondary dormancy acquisition of B. tectorum. In the laboratory approximately 80% of seeds incubated at –1.0 MPa became dormant. In the field, seeds were placed in the seed bank in late autumn, retrieved monthly and dormancy status determined. The field study confirmed the laboratory results; ungerminated seeds became increasingly dormant. Our data suggest that secondary dormancy is much more likely to occur at xeric sites.