Suzette Clement

The quick and the deadly: growth vs virulence in a seed bank pathogen

*We studied the relationship between virulence (ability to kill nondormant Bromus tectorum seeds) and mycelial growth index in the necrotrophic seed pathogen Pyrenophora semeniperda. Seed pathosystems involving necrotrophs differ from those commonly treated in traditional evolution-of-virulence models in that host death increases pathogen fitness by preventing germination, thereby increasing available resources. Because fast-germinating, nondormant B. tectorum seeds commonly escape mortality, we expected virulence to be positively correlated with mycelial growth index. *We performed seed inoculations using conidia from 78 pathogen isolates and scored subsequent mortality. For a subset of 40 of these isolates, representing a range of virulence phenotypes, we measured mycelial growth index. *Virulence varied over a wide range (3-43% seed mortality) and was significantly negatively correlated with mycelial growth index (R(2) = 0.632). More virulent isolates grew more slowly than less virulent isolates. *We concluded that there is an apparent tradeoff between virulence and growth in this pathogen, probably because the production of toxins necessary for necrotrophic pathogenesis competes with metabolic processes associated with growth. Variation in both virulence and growth rate in this pathosystem may be maintained in part by seasonal variation in the relative abundance of rapidly germinating vs dormant host seeds available to the pathogen.

Pyrenophoric Acids B and C, Two New Phytotoxic Sesquiterpenoids Produced by 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.

Ecological genetics of the Bromus tectorum (Poaceae)-Ustilago bullata (Ustilaginaceae) pathosystem: A role for frequency-dependent selection?

UNLABELLED PREMISE OF THE STUDY Evolutionary processes that maintain genetic diversity in plants are likely to include selection imposed by pathogens. Negative frequency-dependent selection is a mechanism for maintenance of resistance polymorphism in plant-pathogen interactions. We explored whether such selection operates in the Bromus tectorum-Ustilago bullata pathosystem. Gene-for-gene relationships between resistance and avirulence loci have been demonstrated for this pathosystem. • METHODS We used molecular markers and cross-inoculation trials to learn whether the SSR genotypes of the host exhibited resistance to co-occurring pathogen races, whether host genotypes within a population had equal disease probability, and whether a common resistance locus and its corresponding avirulence locus exhibited predicted allele frequency changes during an epidemic. • KEY RESULTS Five of six putative resistance loci that conferred resistance to co-occurring pathogen races occurred in common host SSR genotypes. Some common genotypes within populations were more likely to be diseased than others, and genotype frequencies sometimes changed across years in patterns consistent with frequency-dependent selection. Observed changes in frequency of resistance and virulence alleles during an epidemic provided further support, but evidence was inconclusive. • CONCLUSIONS Frequency-dependent selection may operate at endemic disease levels in this pathosystem, but is difficult to detect because many susceptible plants escape infection. Most pathogen isolates were virulent on most host genotypes, minimizing the apparent importance of frequency-dependent selection even during epidemics.

Genetic Variation in Ustilago bullata: Molecular Genetic Markers and Virulence on Bromus tectorum Host Lines

The pathogen Ustilago bullata often causes epidemic levels of head smut disease in Intermountain populations of the inbreeding annual grass Bromus tectorum. We examined patterns of genetic variation for virulence and for amplified fragment length polymorphism (AFLP) markers in four U. bullata populations on B. tectorum in northern Utah and southern Nevada. Patterns of disease incidence generally supported a gene‐for‐gene model for virulence and resistance in this pathosystem. Most host lines were susceptible to most or all paired isolates included in cross‐inoculation tests, but we found evidence for the existence of four avirulence genes. Host lines with alleles conferring resistance were usually resistant to races not found in co‐occurring pathogen populations. The exception was the population at Potosi Pass, Nevada, where the prevalent host line possessed an allele conferring resistance to one of two co‐occurring races. The two pathogen races at Potosi Pass were also strongly genetically differentiated in terms of their AFLP genotypes, whereas for the other three populations, there was no relationship between virulence phenotype and AFLP genotype. There was little evidence for high levels of inbreeding in the northern pathogen populations, which showed no clear groupings of isolates within or among populations. The correlation of the virulence phenotype with the AFLP genotype at Potosi Pass was associated with the presence of a mating locus–linked haplolethal trait in the race pathogenic on the prevalent host line. The presence of this trait is coincident with a high level of intratetrad selfing in this race, thus isolating it from the co‐occurring race and leading to fixation of most AFLP markers. The Potosi Pass population was also clearly differentiated from the northern populations, which were genetically similar to each other, a pattern also seen in the co‐occurring host populations, providing evidence for the idea of pathogen dispersal through seedborne inoculum.

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.

Cochliotoxin, a Dihydropyranopyran-4,5-dione, and Its Analogues Produced by Cochliobolus australiensis Display Phytotoxic Activity against Buffelgrass (Cenchrus ciliaris)

Buffelgrass (Pennisetum ciliare or Cenchrus ciliaris) is a perennial grass that has become highly invasive in the Sonoran Desert of southern Arizona. In the search for novel control strategies against this weed, strains of the foliar fungal pathogen Cochliobolus australiensis from buffelgrass have been screened for their ability to produce phytotoxic metabolites that could potentially be used as natural herbicides in an integrated pest management strategy. A new phytotoxin, named cochliotoxin, was isolated from liquid culture of this fungus together with radicinin, radicinol, and their 3-epimers. Cochliotoxin was characterized, essentially by spectroscopic methods, as 3-hydroxy-2-methyl-7-(3-methyloxiranyl)-2,3-dihydropyrano[4,3-b]pyran-4,5-dione. Its relative stereochemistry was assigned by 1H NMR techniques, while the absolute configuration (2S,3S) was determined applying the advanced Moshers method by esterification of its hydroxy group at C-3. When bioassayed in a buffelgrass coleoptile elongation test and by leaf puncture bioassay against the host weed and two nontarget grasses, cochliotoxin showed strong phytotoxicity. In the same tests, radicinin and 3-epi-radicinin also showed phytotoxic activity, while radicinol and 3-epi-radicinol were largely inactive. All five compounds were more active in leaf puncture bioassays on buffelgrass than on the nontarget grass tanglehead (Heteropogon contortus), while the nontarget grass Arizona cottontop (Digitaria californica) was more sensitive to radicinin and 3-epi-radicinin. Cochliotoxin at low concentration was significantly more active on buffelgrass than on either native grass, but the difference was small.

Phytotoxic activity against Bromus tectorum for secondary metabolites of a seed-pathogenic Fusarium strain belonging to the F. tricinctum species complex

Abstract The winter annual grass Bromus tectorum (cheatgrass) has become highly invasive in semiarid ecosystems of western North America. In these areas, a natural phenomenon, complete cheatgrass stand failure (‘die-off’), is apparently caused by a complex interaction among soilborne fungal pathogens. Several Fusarium strains belonging to the Fusarium tricinctum species complex were isolated from these soils and found to be pathogenic on B. tectorum seeds. One of these strains was produced in cheatgrass seed culture to evaluate its ability to produce phytotoxins. Six metabolites were isolated and identified by spectroscopic methods (essentially 1D and 2D NMR and ESIMS) as acuminatopyrone (1), blumenol A (2), chlamydosporol (3), isochlamydosporol (4), ergosterol (5) and 4-hydroxybenzaldehyde (6). Upon testing against B. tectorum in a seedling bioassay, (6) the coleoptile and radicle length of cheatgrass seedlings were significantly reduced. Compounds 1 and 2 showed moderate activity, while 3–5 were not significantly different from the control.

Chloromonilinic Acids C and D, Phytotoxic Tetrasubstituted 3-Chromanonacrylic Acids Isolated from Cochliobolus australiensis with Potential Herbicidal Activity against Buffelgrass (Cenchrus ciliaris)

The fungal pathogen Cochliobolus australiensis isolated from infected leaves of the invasive weed buffelgrass (Pennisetum ciliare) was grown in vitro to evaluate its ability to produce phytotoxic metabolites that could potentially be used as natural herbicides against this weed. Two new tetrasubstituted 3-chromanonacrylic acids, named chloromonilinic acids C (1) and D (2), were isolated from the liquid cultures of C. australiensis, together with the known chloromonilinic acid B. Chloromonilinic acids C and D were characterized by spectroscopic and chemical methods as (E)-3-chloro-3-[(5-hydroxy-3-(1-hydroxy-2-methoxy-2-oxoethyl)-7-methyl-4-oxo-4H-chromen-2-yl)]acrylic acid and (Z)-3-chloro-3-[(5-hydroxy-3-(2-methoxy-2-oxoethyl)-7-methyl-4-oxo-4H-chromen-2-yl)]acrylic acid, respectively. The stereochemistry of chloromonilinic acids C and D was determined using a combination of spectroscopic and computational methods, including electronic circular dichroism. The fungus produced these compounds in two different liquid media together with cochliotoxin, radicinin, radicinol, and their 3-epimers. The radicinin-related compounds were also produced when the fungus was grown in wheat seed solid culture, but chloromonilinic acids were not found in the solid culture organic extract. All three chloromonilinic acids were toxic to buffelgrass in a seedling elongation bioassay, with significantly delayed germination and dramatically reduced radicle growth, especially at a concentration of 5 × 10-3 M.

Pyriculins A and B, two monosubstituted hex‐4‐ene‐2,3‐diols and other phytotoxic metabolites produced by Pyricularia grisea isolated from buffelgrass (Cenchrus ciliaris)

Pyricularia grisea has been identified as a foliar pathogen on buffelgrass (Cenchrus ciliaris) in North America and was studied as a potential source of phytotoxins for buffelgrass control. Two monosubstituted hex-4-ene-2,3-diols, named pyriculins A and B, were isolated from its culture filtrate organic extract together with (10S,11S)-(-)-epipyriculol, trans-3,4-dihydro-3,4,8-trihydroxy-1(2H)-napthalenone, and (4S)-(+)-isosclerone. Pyriculins A and B were characterized by spectroscopic (essentially nuclear magnetic resonance [NMR], High-resolution electrospray ionization mass spectrometry [HRESIMS]) and chemical methods such as (4E)-1-(4-hydroxy-1,3-dihydroisobenzofuran-1-yl)hex-4-ene-2,3-diols. The relative and absolute configuration of these compounds was determined by a combination of spectroscopic (NMR, electronic circular dichroism [ECD]) and computational tools. When bioassayed in a buffelgrass coleoptile and radicle elongation test, (10S,11S)-(-)-epipyriculol proved to be the most toxic compound. Seed germination was much reduced and slowed with respect to the control and radicles failed to elongate. All five compounds delayed germination, but only (10S,11S)-(-)-epipyriculol was able to prevent radicle development of buffelgrass seedlings. It had no effect on coleoptile elongation, while the other four compounds caused significantly increased coleoptile development relative to the control.

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.