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Radicinin is a phytotoxic fungal dihydropyranopyran-4,5-dione under evaluation for the development of a target-specific bioherbicide for invasive buffelgrass (Cenchrus ciliaris) control. It has already demonstrated high toxicity on host plants, low toxicity to native plants and no negative effects on zebrafish embryos. To continue these studies at the whole-plant level there is a need to obtain much larger quantities of radicinin, either by optimizing its large-scale production by fungal fermentation or through its total stereoselective synthesis. A rapid and sensitive HPLC method for quantification of radicinin in complex mixtures has been developed in order to evaluate its production by different Cochliobolus australiensis strains and in different cultural conditions. The analysis proved that radicinin is not produced by all the strains tested and its synthesis is strongly affected by cultural conditions. The HPLC method could be useful in selecting the best fungal source for the production of this promising potential bioherbicide.

The fungal pathogen Pyricularia grisea has been studied to evaluate its production of phytotoxins for the biocontrol of the buffelgrass (Cenchrus ciliaris L.) weed. A first investigation allowed to isolate several new and known phytotoxic metabolites. However, the further investigation on the organic extract obtained from the fungus liquid culture showed the presence of other metabolites possibly contributing to its phytotoxicity. Thus, four known metabolites were isolated and identified by spectroscopic (nuclear magnetic resonance [NMR] and high-resolution electrospray ionization mass spectrometry [HRESIMS]) methods as dihydropyriculol (1), epi-dihydropyriculol (2), 3-methoxy-6,8-dihydroxy-3-methyl-3,4-dihydroisocoumarin (3), and (R)-mevalonolactone (4). The absolute configuration of 1-3 was determined for the first time by a computational analysis of their electronic circular dichroism (ECD) spectra. When the isolated compounds were bioassayed at a concentration of 5 × 10-3 M in a buffelgrass coleoptile and radicle elongation test no toxicity was detected. On the contrary, compounds 1 and 3 showed a significant stimulating effect of radical elongation. Furthermore, the difference in growth stimulation between 1 and its epimer 2 highlights the tight relationship between absolute configuration and biological activity of these fungal metabolites.

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.

Radicinin is a phytotoxic fungal dihydropyranopyran-4,5-dione under evaluation for the development of a target-specific bioherbicide for invasive buffelgrass (

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., The fungus Pyrenophora semeniperda produces pyrenophoric acid B, a small molecule that exploits the plant ABA biosynthetic pathway to reduce seed germination, increasing its reproductive success.

Radicinin (1), is a fungal dihydropyranopyran-4,5-dione isolated together with some analogues, namely 3-epi-radicinin, radicinol, 3-epi-radicinol, and cochliotoxin (2&ndash, 5), from the culture filtrates of the fungus Cochliobolus australiensis, a foliar pathogen of buffelgrass (Cenchrus ciliaris), an invasive weed in North America. Among the different metabolites 1 showed target-specific activity against the host plant and no toxicity on zebrafish embryos, promoting its potential use to develop a natural bioherbicide formulation to manage buffelgrass. These data and the peculiar structural feature of 1 suggested to carry out a structure-activity relationship study, preparing some key hemisynthetic derivatives and to test their phytotoxicity. In particular, p-bromobenzoyl, 5-azidopentanoyl, stearoyl, mesyl and acetyl esters of radicinin were semisynthesized as well as the monoacetyl ester of 3-epi-radicinin, the diacetyl esters of radicinol and its 3 epimer, and two hexa-hydro derivatives of radicinin. The spectroscopic characterization and the activity by leaf puncture bioassay against buffelgrass of all the derivatives is reported. Most of the compounds showed phytotoxicity but none of them had comparable or higher activity than radicinin. Thus, the presence of an &alpha, &beta, unsaturated carbonyl group at C-4, as well as, the presence of a free secondary hydroxyl group at C-3 and the stereochemistry of the same carbon proved to be the essential feature for activity.

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 Mosher's 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. © 2017 The American Chemical Society and American Society of Pharmacognosy.

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.

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.

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.

A novel spirocyclic γ-lactam, named spirostaphylotrichin W ( 1 ), was isolated together with the well known and closely related spirostaphylotrichins A, C, D, R and V, as well as triticone E, from the liquid cultures of Pyrenophora semeniperda (anamorph: Drechslera ), a seed pathogen proposed for cheatgrass ( Bromus tectorum ) biocontrol. Spirostaphylotrichin W was characterized as (3 S *,4 S *,5 S *,6 S *,9 Z ,10 Z )-4,6-dihydroxy-2,3-dimethoxy-3-methyl-10-propyliden-2-azaspiro [4.5]dec-8-ene-1,7-dione, by spectroscopic and chemical methods. The relative stereochemistry of spirostaphylotrichin W was assigned using NOESY experiments and in comparison to those of spirostaphylotrichin V ( 2 ) and triticone E ( 6 ). In fact, the relative stereochemistry at C-3 was the same of that of 2 , while that at C-4 and C-6 was inverted in respect to that reported, respectively, for 2 and 6 . In a B. tectorum coleoptile bioassay at concentration of 10 −3 , spirostaphylotrichin A proved to be the most active compound, followed by spirostaphylotrichins C and D. Spirostaphylotrichin W and V showed mild toxicity while spirostaphylotrichin R and triticone E were not active. When tested on host and non-host plants by leaf puncture bioassay, spirostaphylotrichins A, C and D caused the appearance of necrotic spots while the other compounds were inactive.

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.

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 9 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.

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...

Bromus tectorum L. (cheatgrass or downy brome) is an important exotic weed in natural ecosystems as well as in winter cereal cropland in semiarid western North America. The systemic, seedling-infecting head smut pathogen Ustilago bullata Berk. commonly infects cheatgrass stands, often at epidemic levels. We examined factors controlling U. bullata infection levels in greenhouse studies with parental lines of four B. tectorum populations from contrasting habitats and U. bullata bulk teliospore collections from within the four populations. The U. bullata infection process appeared to have broad environmental tolerances, so that it was relatively simple to develop a protocol for obtaining high infection percentages in susceptible lines. Bromus tectorum populations generally showed highest infection levels when inoculated with locally collected U. bullata teliospores. This effect was most marked for the warm desert population, which was completely resistant to U. bullata collected from other areas, but 100% su...

 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 diffi cult 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.

Summary • 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.