Non-indolyl cruciferous phytoalexins: Nasturlexins and tridentatols, a striking convergent evolution of defenses in terrestrial plants and marine animals?

Phytochemistry xxx (2014) xxx–xxx

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The first non-indolyl cruciferous phytoalexins: Nasturlexins and tridentatols, a striking convergent evolution of defenses in terrestrial plants and marine animals? M. Soledade C. Pedras ⇑, Q. Huy To Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK S7N 5C9, Canada

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Article history: Available online xxxx Keywords: Alternaria brassicicola Brassicaceae Brassinin Crucifer Gluconasturtiin Leptosphaeria maculans Nasturlexin Nasturtium officinale Phytoalexin Sclerotinia sclerotiorum Tridentata marginata

a b s t r a c t Highly specialized chemical defense pathways are a particularly noteworthy metabolic characteristic of sessile organisms, whether terrestrial or marine, providing protection against pests and diseases. For this reason, knowledge of the metabolites involved in these processes is crucial to producing ecologically fit crops. Toward this end, the elicited chemical defenses of the crucifer watercress (Nasturtium officinale R. Br.), i.e. phytoalexins, were investigated and are reported. Almost three decades after publication of cruciferous phytoalexins derived from (S)-Trp, phytoalexins derived from other aromatic amino acids were isolated; their chemical structures were determined by analyses of their spectroscopic data and confirmed by synthesis. Nasturlexin A, nasturlexin B, and tridentatol C are hitherto unknown phenyl containing cruciferous phytoalexins produced by watercress under abiotic stress; tridentatol C is also produced by a marine animal (Tridentata marginata), where it functions in chemical defense against predators. The biosynthesis of these metabolites in both a terrestrial plant and a marine animal suggests a convergent evolution of unique metabolic pathways recruited for defense. Ó 2014 Published by Elsevier Ltd.

1. Introduction The immense biodiversity encountered on Earth is associated with the biosynthesis of numerous and structurally different secondary metabolites common to phylogenetically related groups. In spite of this, examples of convergent evolution are starting to reveal that a few specialized metabolites are common to plants, animals and microorganisms (Pichersky and Lewinsohn, 2011). Perhaps one of the most striking examples of metabolic convergent evolution is the gibberellin biosynthetic pathway, as these metabolites are produced by higher plants, fungi, and bacteria (Fischbach and Clardy, 2007). Similarly, cyanogenic glucosides, metabolites that have strong defensive roles in plants, are also produced in insect larva via convergent evolution of pathways (Jensen et al., 2011). By contrast, co-occurrence of identical or similar secondary metabolites in terrestrial and marine organisms is far less common (Cutignano et al., 2012; Sy-Cordero et al., 2011; Daly, 2004). Crucifers (plant family Brassicaceae) contain numerous species of global economic importance due to the oil content of seeds (canola, rapeseed, mustard) and the great nutritional and flavoring

⇑ Corresponding author. Tel.: +1 (306)966 4772; fax: +1 (306)966 4730. E-mail address: [email protected] (M.S.C. Pedras).

value of whole plants (e.g. cabbage, broccoli, rutabaga, cauliflower, Brussels sprouts, radish, wasabi, mustard) (Warwick, 2011; Gupta, 2009). In addition, Arabidopsis thaliana (http://www.arabidopsis.org/) and Thellungiella salsuginea (http://thellungiella.org/literature.php) are widely investigated crucifers due to their great scientific importance as model species. Watercress (Nasturtium officinale R. Br.) is also an edible crucifer widely used in salads due to its peppery flavor and easy cultivation. Chemical analysis revealed it is a healthy source of vitamins, anti-carcinogens (Palaniswamy et al., 2003) and anti-inflammatory phytochemicals (Sadeghi et al., 2014). Our longstanding interest in the chemical defenses of crucifers, particularly phytoalexins, is directed to uncover metabolic pathways that might protect these crops from microbial diseases (Pedras and Yaya, 2010; Pedras et al., 2011). Phytoalexins are secondary metabolites with antimicrobial activity, produced in response to stress such as pathogen attack, heavy metal salts, UV radiation, but not present in naturally healthy plants (Bailey and Mansfield, 1982; Mansfield and Bailey, 1982; VanEtten et al., 1994). Their chemical structures within a given phylogenetic group derive from common precursor(s) and biosynthetically interconnected pathways. Access to sources of structurally diverse phytoalexins provide great prospects to generate crops having high resistance levels to fungal pathogens and abiotic stress (Grosskinsky et al., 2012).

http://dx.doi.org/10.1016/j.phytochem.2014.07.024 0031-9422/Ó 2014 Published by Elsevier Ltd.

Please cite this article in press as: Pedras, M.S.C., To, Q.H. The first non-indolyl cruciferous phytoalexins: Nasturlexins and tridentatols, a striking convergent evolution of defenses in terrestrial plants and marine animals? Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.07.024

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M. Soledade C. Pedras, Q.H. To / Phytochemistry xxx (2014) xxx–xxx

Fig. 1. Biosynthetic relationships of (S)-tryptophan (Trp), glucobrassicins (1–1b) and phytoalexins of scaffold A, brassinins (5–5b), and scaffold B, rapalexins (7).

As summarized in Fig. 1, the cruciferous phytoalexins identified to date are biosynthetically derived from (S)-tryptophan (Trp), in most cases via indole glucosinolates named glucobrassicins (1– 1b) (Pedras et al., 2011) after enzymatic hydrolyses. Brassinins (5–5b) and related phytoalexins containing type A scaffold were first reported in 1986 (Takasugi et al., 1986), whereas phytoalexins of type B scaffold (isalexin, rapalexins, isocyalexin A) were reported much later (Pedras et al., 2004b, 2007; Pedras and Yaya, 2012). Glucosinolates are a broad class of secondary metabolites that upon enzymatic hydrolyses (EC 3.2.1.147 – thioglucosidases, myrosinases) yield various products including isothiocyanates; both glucosinolates and isothiocyanates have very important ecological roles in crucifers (Agerbirk and Olsen, 2012). Indolyl-3methylisothiocyanates (3) are chemically very reactive and thus are not naturally occurring (Agerbirk et al., 2009), but indole-3-isothiocyanates like rapalexin A (7, R@H; R0 @OMe) are stable (Fig. 1) and strongly antifungal (Pedras et al., 2011). It was hypothesized that benzylisothiocyanates and homologues may be potential precursors of different phytoalexins (Pedras and Smith, 1997) via pathways similar to that highlighted for brassinins (5–5b) (Fig. 1). For example, white mustard (Sinapis alba L.) that produces glucosinalbin (8) and the corresponding phydroxybenzylisothiocyanate (9) (Du et al., 1995) might produce phenyl-containing phytoalexins. Nonetheless, a search for phytoalexins produced by S. alba disclosed only metabolites derived from glucobrassicins (1–1b) (Pedras and Smith, 1997; Pedras and Zaharia, 2000). Finally, almost three decades after publication of the first phytoalexins derived from (S)-Trp, cruciferous phytoalexins were uncovered that are derived from other aromatic amino acids: nasturlexin A (10), nasturlexin B (11), and tridentatol C (12). Unexpectedly, one of the new phytoalexins (tridentatol C (12)) is also produced by a marine animal (Tridentata marginata) where,

together with related metabolites, it functions as chemical defense against predators (Lindquist et al., 1996). The biosynthesis of these metabolites in both a terrestrial plant and a marine animal suggests a convergent evolution of unique metabolic pathways recruited for defense. The discovery of new and potent cruciferous defenses could facilitate the design and development of unique ‘‘phytoalexin designer crops’’ with ecologically desirable disease resistance levels. 2. Results and discussion 2.1. Isolation and structure determination of elicited metabolites 10– 12 and phenylethyl isothiocyanate (15) Watercress plants were elicited, incubated, the leaves harvested, then immediately frozen in liquid nitrogen, and extracted with organic solvents, as described in the Experimental. Control plants (non-elicited) were treated similarly. HPLC-DAD-ESI-MS analyses of time-course experiments established that strong elicitation occurred after 24 h of incubation (Table S1, Supplementary Data). The HPLC chromatograms of elicited leaf extracts displayed a readily identifiable component with tR = 18.0 min available in our HPLC-DAD-MS libraries (Pedras et al., 2006, 2008), corresponding to brassinin (5), and four peaks at tR = 11.5, 12.4, 13.8 and 21.5 min due to unknown metabolites (Fig. 2). Three of the elicited compounds were isolated from the EtOAc extract of a larger scale experiment and separated using column chromatography as summarized in the Experimental and detailed in Supporting Data (Fig. S3). The metabolite with tR = 12.4 min could not be purified as it appeared to decompose during the first chromatographic step. The 1H NMR spectroscopic data of brassinin (5) confirmed the structure suggested by the HPLC-DAD-ESI-MS data. The HRMS-EI data of nasturlexin A (10), tR = 21.5 min, suggested a molecular



A

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B

Fig. 2. HPLC-DAD chromatograms of extracts of leaves of watercress (detection 220 nm): (A) CuCl2 elicited plants, 24 h incubation; (B) control plants, 24 h incubation. Peaks of metabolites identified after isolation: tR = 11.5 min, nasturlexin B (11); tR = 13.8 min, tridentatol C (12); tR = 18.0 min, brassinin (5); tR = 21.5, nasturlexin A (10); tR = 24.7 min, phenylethyl isothiocyanate (15).

formula of C10H13NS2 that indicated five degrees of unsaturation; the 1H NMR spectroscopic data suggested a phenylethyl moiety and an S-methyl substituent. The HRMS-EI data of nasturlexin B (11), tR = 11.5 min, suggested a molecular formula of C10H11NOS2, and a structure containing six degrees of unsaturation. The 1H NMR data also suggested a p-hydroxyphenyl attached to a spin system of three protons plus an S-methyl substituent. The third metabolite (12) with tR = 13.8 min had a molecular formula of C10H9NOS2, indicating a structure containing seven degrees of unsaturation; its 1H NMR data suggested a p-hydroxyphenyl attached to a thiazolyl ring with a S-methyl substituent. Based on these data, structures 10–12 were proposed, a literature search indicated that compound 12 had been previously isolated from the marine hydroid T. marginata (tridentatol C (12)) (Lindquist et al., 1996), while compounds 10 and 11 had not been previously obtained from natural sources. The three compounds were synthesized as reported in Supporting Data and their spectroscopic data was determined to be identical to the natural products isolated from stressed watercress. Hence, the structural assignments of the new metabolites obtained from elicited watercress, nasturlexins A (10) and B (11) and tridentatol C (12), were confirmed unambiguously. The value of the specific optical rotation of natural nasturlexin B (11) was close to zero, suggesting that the compound was not enantiomerically pure. Further analysis of the synthetic material and the natural product using chiral HPLC indicated a racemic mixture in both cases. Although not expected, it is likely that a potential cyclizing enzyme involved in the biosynthesis of 11 is not stereospecific, as in a few other instances (Pedras et al., 2004a; Batista et al., 2009; Vallakati et al., 2013). HPLC analysis of the hexane extract of the large-scale experiment of elicited leaves described in Section 4.4 showed the presence of a constituent with tR = 24.7 min also present in control leaves (Fig. 2B). This metabolite was separated by chromatography (6 mg) and was characterized by NMR, UV and HRMS spectroscopic data (Supplementary Data). The structure of this metabolite was assigned as phenylethyl isothiocyanate (15) and was consistent with previously published data (Lim et al., 2008). By analogy to the brassinins and rapalexins pathway (Fig. 1), nasturlexins A (10) and B (11) and tridentatol C (12) are likely to be biosynthetically derived from gluconasturtiin (14), via phenylethyl isothiocyanate (15), which upon elicitation would be thiomethylated by an inducible enzyme (Fig. 3). The glucosinolate gluconasturtiin (14) is produced by watercress in higher amounts than in other cruciferous species (Kiddle et al., 2001) from the amino acid precursor homophenylalanine (13) (Dawson et al.,

1993; Du et al., 1995; Sønderby et al., 2010). Alternatively, nasturlexin B (11) and tridentatol C (12) might be biosynthetically derived from p-hydroxygluconasturtiin (14a), via p-hydroxyphenylethyl isothiocyanate (15a). Occurrence of p-hydroxygluconasturtiin (14a) has been reported in Arabis hirsuta (L.) Scop. and Arabis soyeri Reut. & Huet subsp. subcoriacea (Gren. ex Nyman) Breitstr. (Brassicaceae) (Agerbirk et al., 2010) and is likely to be produced in several other crucifer species. However, it remains to be determined if 14a is biosynthesized from Phe or Tyr. On the other hand, since marine organisms do not appear to produce glucosinolates, it is likely that in T. marginata the biosynthetic pathways of tridentatols like 17, 17a and 18 (Lindquist, 2002) involve other intermediates in which the isothiocyanate carbon derives from an undetermined one-carbon donor. Likewise, coriandrins A (16) and B (16a) and p-methoxytridentatols A (17b) and B (17c) produced by liverworts (Corsinia coriandrina, Hepaticae) (Reuß and König, 2005), use a biosynthetic pathway derived from Tyr, similar to tridentatols (Reuß, 2009). 2.2. Antifungal activity of metabolites Having reasonable amounts of elicited compounds 10–12 and constitutive phenylethyl isothiocyanate (15) in hand allowed the determination of their antifungal activities against economically important fungal pathogens of crucifers (Table 1). Control plates contained PDA medium and 1% DMSO, the solvent used to dissolve tested compounds, as described in the Experimental. The specific fungal species Alternaria brassicicola (Schwein.) Wiltshire and Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.], which infect crucifers, specially Brassica species, and the generalist plant pathogen Sclerotinia sclerotiorum (Lib.) de Bary that infects various plant families including crucifers were used (Pedras et al., 2011). As summarized in Table 1, tridentatol C (12) was the most inhibitory of the four compounds tested, causing complete inhibition of mycelial growth in the three species at 0.50 mM, whereas nasturlexin A (10) caused complete inhibition of mycelial growth of L. maculans and S. sclerotiorum. Interestingly, S. sclerotiorum seemed to be the most sensitive of the three fungal species, particularly to phenylethyl isothiocyanate (15) (complete inhibition at 0.20 mM). Considering that compounds 10–12 are antifungal and inducible metabolites produced in stressed watercress leaves, they are phytoalexins, whereas 15 is a constitutive metabolite thus should be considered a phytoanticipin. Lastly, comparison of the concentrations of nasturlexin B (11) (164 ± 36 nmol/ g fresh wt) and tridentatol C (12) (142 ± 32 nmol/g fresh wt)


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Fig. 3. Biosynthetic relationships of phenylalanine (Phe), tyrosine (Tyr), homophenylalanine (13), gluconasturtiin (14), phenylethyl isothiocyanate (15) nasturlexins A (10) and B (11) and coriandrins (16) and tridentatols A (17), B (17a), C (12) and D (18), and p-methoxytridentatols A (17b), B (17c); N. o. = Nasturtium officinale, T. m. = Tridentata marginata and C. c. = Corsinia coriandrina; dashed arrows are proposed biosynthetic steps. Table 1 Antifungal activity of metabolites of watercress (Nasturtium officinale) against fungal pathogens: Alternaria brassicicola, Leptosphaeria maculans and Sclerotinia sclerotiorum (20– 130 h incubation on agar plates). Compound (mM)

Nasturlexin A (10) 0.50 0.20 0.10 Nasturlexin B (11) 0.50 0.20 0.10 Tridentatol C (12) 0.50 0.20 0.10 Phenylethylisothiocyanate (15) 0.50 0.20 0.10

% Inhibitiona ± Standard deviation A. brassicicolab

L. maculansc

S. sclerotiorumd

69 ± 5 44 ± 5 27 ± 3

Complete inhibition 84 ± 3 29 ± 5


76 ± 5 43 ± 4 20 ± 5

94 ± 0 16 ± 3 9±3

59 ± 8 27 ± 8 13 ± 6




No inhibition No inhibition No inhibition

68 ± 8 20 ± 0 8±3

Complete inhibition Complete inhibition 68 ± 8

a Percentage of growth inhibition calculated using the formula:% inhibition = 100 – [(growth on amended/growth in control) 100]; values represent the mean and standard deviation of at least two independent experiments conducted in triplicate. Control plates contained PDA medium and 1% DMSO, the solvent used to dissolve tested compounds, as described in the Experimental. b Incubated for 70 h under continuous light. c Incubated for 130 h under continuous light. d Incubated for 20 h in the dark.

present in leaves of watercress two days after elicitation with CuCl2 (Table S1) and their antifungal activity (Table 1), and considering that both compounds are produced simultaneously, suggests that the plant biosynthesizes both compounds in sufficient amounts to inhibit the growth of the three fungal species tested. 3. Conclusion Watercress under abiotic stress produces two structural groups of phytoalexins derived from different aromatic amino acids:

nasturlexins A (10) and B (11), and tridentatol C (12) likely derived from Phe via gluconasturtiin (14), and brassinin (5) derived from Trp via glucobrassicin (1). This is the first crucifer reported to synthesize phytoalexins from parallel pathways, and, to the best of our knowledge, the first example of a phytoalexin also produced in a marine animal. These findings suggest a convergent evolution of unique metabolic pathways recruited for defense in both terrestrial plants and marine animals. Further work needs to be carried out to determine if watercress produces additional phytoalexins under different stress conditions. In addition, the intermediates



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a mass detector (Agilent G2440A MSD-Trap-XCT ion trap mass spectrometer) with an electrospray ionization (ESI) source. Chromatographic separations were carried out at room temperature using an Eclipse XDB-C-18 column (5 lm particle size silica, 150 mm 4.6 mm I.D.). The mobile phase (method B) consisted of a linear gradient of H2O (with 0.2% HCO2H) – CH3CN (with 0.2% HCO2H) from 75:25 to 25:75 in 25 min and a flow rate of 1.0 mL/min. Data acquisition was carried out in positive and negative polarity modes in a single LC run, and data processing carried out with Agilent Chemstation Software. 4.3. Plant material and extractions

of the biosynthetic pathway of nasturlexins need to be established. If the biosynthesis occurs as proposed above, it is likely that only a few additional steps would be required to engineer Brassica species to biosynthesize nasturlexins. Finally, it is conceivable that species related to N. officinale might provide a wider range of phytoalexins useful to develop cruciferous crops potentially more resistant to fungal diseases.

Seeds of watercress were obtained from a commercial source (Sand Mountain Herbs, www.sandmountainherbs.com) and sown in a perlite and nutrient free LG-3 soil (Sun Gro Horticulture Canada) in small pots in a growth chamber at 16 h of light/8 h of dark, 20 °C day/18 °C night, 350–360 lE m2 s1, and with ambient humidity. For elicitation of phytoalexins, 4-week-old plants were sprayed with a CuCl2 aq. solution (2 mM) and plants were kept in the growth chamber. Control leaves were sprayed with H2O. The leaves (ca. 1.5 g fresh weight per sample) were excised after 1, 2, 4, 6 days after elicitation, were frozen in liq. N2, ground and the resulting leaf materials were individually extracted with MeOH (5 mL). The extracts were filtered, the filtrates were concentrated and rinsed with CH2Cl2. The CH2Cl2 extracts were concentrated, dissolved in CH3CN–MeOH (1:1) and analyzed by HPLC-DAD using method A and by HPLC-DAD-ESI-MS using method B. For isolation of the unknown metabolites detected in elicited extracts, a larger scale experiment was carried out and leaves were extracted as follows.

4. Experimental 4.4. Compound isolation, synthesis and spectroscopic characterization 4.1. General experimental All solvents were HPLC grade and used as such, except for those used in chemical syntheses, as noted. Unless otherwise noted, materials were obtained from commercial suppliers and used without further purification. Flash column chromatography (FCC): silica gel, grade 60, 230–400 lm. Organic extracts were dried over Na2SO4 and the solvents were removed using a rotary evaporator. NMR spectra were recorded on Bruker Avance 500 MHz spectrometers. For 1H NMR (500 MHz) and 13C NMR (125.8 MHz) spectra, the chemical shifts (d) are reported in parts per million (ppm) relative to TMS. 4.2. HPLC analyses HPLC-DAD analysis was carried out with Agilent 1100 and 1200 series systems all equipped with quaternary pump, autosampler, diode array detector (DAD, wavelength range 190–600 nm), and degasser. Method A (phytoalexins and non-polar metabolites) used a Zorbax Eclipse XDB-C18 column (5 lm particle size silica, 150 4.6 mm I.D.), equipped with an in-line filter, with the mobile phase H2O–CH3CN from 75:25 to 25:75, linear gradient for 35 min, and a flow rate of 1.0 mL/min. Chiral HPLC analysis was carried out with an Agilent 1100 system equipped with a quaternary pump, autosampler, UV detector (wavelength 210, 230 nm), and degasser. An IA Chiralpak column (5 lm particle size silica, 2.1 150 mm I.D.), and the mobile phase iPrOH – hexane (1:9, isocratic elution) with a flow rate of 0.10 mL/ min was used to obtain clear separation of both enantiomers. HPLC-DAD-ESI-MS analysis was carried out with an Agilent 1100 series HPLC system equipped with an autosampler, binary pump, degasser, and a diode array detector connected directly to

After incubation for 1 day in a growth chamber, elicited leaves (ca. 155 g) were collected, frozen, ground and rinsed with hexane (2 300 mL). The hexane extract was concentrated (30 mg) and fractionated by flash column chromatography (FCC) to yield phenylethyl isothiocyanate (15) (6.0 mg). The leaf solids were dissolved in MeOH (1 L) and shaken for 1 h. After filtration, the MeOH extract was concentrated and partitioned in H2O-EtOAc and the organic extract was dried and concentrated to give a dark green residue (ca. 1.2 g). After multiple FCCs as described in Supporting Data, nasturlexin A (10) (2.2 mg), brassinin (5) (0.2 mg), nasturlexin B (11) (0.8 mg) and tridentatol C (12) (0.3 mg) were obtained. Nasturlexins A (10) and B (11) and tridentatol C (12) (Jayatilake and Baker, 1999) were synthesized as previously reported and as described in Supplementary Data. Nasturlexin A (10). HPLC tR = 21.5 min (method A). 1H NMR (500 MHz, CDCl3): d 7.34 (2H, t, J = 7.5 Hz), 7.27–7.21 (m, 3H), 6.94 (1H, s), 4.02–3.99 (2H, m), 2.98 (2H, t, J = 7.0 Hz), 2.61 (3H, s) and a minor rotamer at 7.65 (s), 3.73–3.69 (m), 2.96 (t), 2.69 (s). 13C NMR (125 MHz, CDCl3): d 199.4, 138.4, 129.1, 129.0, 127.1, 48.2, 34.5, 18.3. HREI-MS m/z (%): calc. for C10H13NS2: 211.0489, found 211.0493 (74), 163.04 (27), 104.06 (100). UV (HPLC, CH3CN–H2O) kmax (nm): 253, 272. Nasturlexin B (11). HPLC tR = 11.5 min (method A). 1H NMR (500 MHz, CDCl3): d 7.14 (2H, d, J = 8.5 Hz), 6.76 (2H, d, J = 8.5 Hz), 5.03 (1H, dd, J = 8.5, 5.5 Hz), 4.48 (1H, dd, J = 14.5, 8.5 Hz), 4.31 (1H, dd, J = 14.5, 5.5 Hz), 2.57 (3H, s). 13C NMR (125 MHz, CDCl3): d 168.8, 156.3, 132.4, 128.6, 116.0, 71.9, 56.3, 15.7. HREI-MS m/z (%): calc. for C10H11NOS2: 225.0282, found 225.0284 (100), 152.03 (62), 87.01 (78). UV (HPLC, CH3CN–H2O) kmax (nm): 220, 290. Tridentatol C (12). HPLC tR = 13.8 min (method A). 1H NMR (500 MHz, CDCl3): d 7.69 (1H, s), 7.37 (2H, d, J = 8.5 Hz), 6.86 (2H,


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d, J = 8.5 Hz), 5.30 (1H, s), 2.71 (3H, s). 13C NMR (125 MHz, CDCl3): d 164.9, 156.3, 139.1, 136.9, 128.2, 123.8, 116.3, 17.1. HREI-MS m/z (%): calc. for C10H9NOS2: 223.0126, found 223.0120 (100), 190.03 (84). UV (HPLC, CH3CN–H2O) kmax (nm): 315. 4.5. Antifungal bioassays The antifungal activity of compounds against three fungal species was investigated using a mycelial radial growth bioassay (PDA media and DMSO solutions of each compound, 0.50, 0.20 and 0.10 mM; control solutions contained 1% DMSO in PDA media). A. brassicicola isolate UAMH 7474 and L. maculans isolate UAMH 9410 were obtained from the University of Alberta Microfungus Collection and Herbarium and S. sclerotiorum clone #33 was obtained from the AAFC Saskatoon Research Center. Spores of A. brassicicola and L. maculans were spotted onto potato dextrose agar plates (PDA) and allowed to grow for seven days under constant light at 23 ± 1 °C. Similarly, sclerotia of S. sclerotiorum were placed on PDA plates and allowed to germinate and grow mycelia for four days incubated in the dark. Plugs (4 mm) were cut from the edges of mycelia and placed inverted onto 12-well plates containing compounds in DMSO mixed into PDA (Pedras and Zaharia, 2000). Control plates contained PDA and 1% DMSO, the solvent used to dissolve tested compounds. The final concentrations of each phytoalexin in agar varied from 0.10 to 0.50 mM, with a DMSO concentration of 1%. Plates were allowed to grow under constant light/ dark at 23 ± 1 °C up to 130 h, as stated in Table 1; the diameter of the mycelial mat was measured and compared to control mycelia grown on plates containing DMSO, using the formula in Table 1 (the diameter of the mycelial plug was subtracted from the total mycelial growth). Acknowledgments Support for the authors’ work was obtained from the Natural Sciences and Engineering Research Council of Canada (Discovery Research Grant to M.S.C.P.) and the University of Saskatchewan (CGSR graduate scholarship to H.T.). We thank M. Gravel and P. Haghshenas, Department of Chemistry, for chiral HPLC analysis and acknowledge the technical assistance of K. Thoms and K. Brown, Department of Chemistry, in HREI-MS and NMR determinations, respectively. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2014. 07.024. References Agerbirk, N., De Vos, M., Kim, J.H., Jander, G., 2009. Indole glucosinolate breakdown and its biological effects. Phytochem. Rev. 8, 101–120. Agerbirk, N., Olsen, C.E., 2012. Glucosinolate structures in evolution. Phytochemistry 77, 16–45. Agerbirk, N., Olsen, C.E., Poulsen, E., Jacobsen, N., Hansen, P.R., 2010. Complex metabolism of aromatic glucosinolates in Pieris rapae caterpillars involving nitrile formation, hydroxylation, demethylation, sulfation, and host plant dependent carboxylic acid formation. Insect Biochem. Mol. Biol. 40, 126–137. Bailey, J.A., Mansfield, J.W., Eds., 1982. Phytoalexins. Blackie and Son, Glasgow, U.K., 334 pp. Batista Jr., J.M., López, S.N., Mota, J.S., Vanzolini, K.L., Cass, Q.B., Rinaldo, D., Vilegas, W., Bolzani, V.S., Kato, M.J., Furlan, M., 2009. Resolution and absolute configuration assignment of a natural racemic chromane from Peperomia obtusifolia (Piperaceae). Chirality 21, 799–801. Cutignano, A., Villani, G., Fontana, A., 2012. One metabolite, two pathways: convergence of polypropionate biosynthesis in fungi and marine molluscs. Org. Lett. 14, 992–995. Daly, J.W., 2004. Marine toxins and nonmarine toxins: convergence or symbiotic organisms? J. Nat. Prod. 67, 1211–1215.

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