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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Lathyroxins A and B, Phytotoxic Monosubstituted Phenols Isolated from Ascochyta lentis var. lathyri, a Fungal Pathogen of Grass Pea (Lathyrus sativus) Marco Masi,† Paola Nocera,† Angela Boari,‡ Alessio Cimmino,† Maria Chiara Zonno,‡ Alessandro Infantino,§ Maurizio Vurro,‡ and Antonio Evidente*,† †
Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Napoli, Italy ‡ Istituto di Scienze delle Produzioni Alimentari (ISPA), Consiglio Nazionale delle Ricerche, Via Amendola, 122/O, 70126 Bari, Italy § Consiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria (CREA), Centro di Ricerca Difesa e Certificazione, Via C.G. Bertero 22, 00156 Rome, Italy S Supporting Information *
ABSTRACT: Ascochyta lentis var. lathyri has recently been reported to be the causal agent of Ascochyta blight of grass pea (Lathyrus sativus), a disease characterized by the appearance of necrotic lesions of leaves and stems. Considering the novelty of the pathogen and the possible involvement of secondary metabolites in symptom appearance, a study was carried out to ascertain the capability of this fungus to produce bioactive metabolites. Some phytotoxic phenols were isolated from the culture filtrates of the fungus. In particular, two new phytotoxic metabolites, named lathyroxins A and B, were characterized by spectroscopic methods as 4-(2-hydroxy-3,3-dimethoxypropyl)phenol and 3-(4-hydroxyphenyl)propane-1,2-diol, respectively, and the R absolute configuration of C-2 of their 2-dimethoxy- and 2,3-diol-propyl side chain was assigned. Moreover, other well-known fungal metabolites, namely, p-hydroxybenzaldehyde, pmethoxyphenol, and tyrosol, were also identified. Lathyroxins A and B showed interesting phytotoxic properties, being able to cause necrosis on leaves and to inhibit seed germination and rootlet elongation. Moreover, both of the new metabolites had no effect against bacteria, arthropods, and nematodes.
T
L.), and A. lentis (teleomorph Didymella lentis W.J. Kaiser, B.C. Wang & J.D. Rogers) in lentil (Lens culinaris Medik.).5 Several pathogens belonging to the Ascochyta genus proved to produce secondary phytotoxic metabolites involved in the appearance of disease symptoms. Recently a pathogenic strain of A. lentis obtained from lentil was shown to produce a new anthraquinone lentisone, together with other well-known metabolites, namely, pachybasin, tyrosol, and pseurotin A.6 Furthermore, from the solid culture of a strain initially identified as A. lathyri the well-known cytochalasins A and B were isolated.7 Considering the novelty of the pathogen, the lack of information about its eventual capability to produce bioactive secondary metabolites (also useful for characterization purposes), and the recognized extreme capability of the genus Ascochyta to produce active metabolites, this was investigated by our group. This study reports the isolation and the chemical and biological characterization of some new phytotoxic and known metabolites produced by A. lentis var. lathyri and some preliminary information on their biological properties and possible involvement in symptom appearance.
he grass pea (Lathyrus sativus L.) is an important food, feed, and fodder legume crop belonging to the Fabaceae family. Over the past decade it has received increased interest as a plant that is adapted to arid conditions and contains high levels of protein. It is a crop of economic importance in many countries, and it is receiving further attention for its beneficial properties in crop rotations and because it is resistant to several legume diseases.1 Recently, a morphological variant of the fungus Ascochyta lentis Vassiljevsky, named A. lentis var. lathyri, was described as infecting grass pea. This fungus causes necrotic lesions on leaves and stems of grass pea plants. The isolates from grass pea show high genomic identity, successful mating, and very similar metabolic profiles with isolates of A. lentis. However, significant differences between the two isolates were observed in conidial dimensions and morphology and strong host specialization, suggesting that the two taxa are genetically distinct and are therefore likely in the process of speciation.2,3 Ascochyta blights are among the most important plant diseases worldwide.4 Among the legume species, Ascochyta blights are caused by different pathogens. For example, ascochytoses are caused by Ascochyta rabiei (Pass.) Labr. (teleomorph Didymella rabiei (Kovatsch.) Arx) in chickpea (Cicer arietinum L.), Ascochyta fabae Speg. (teleomorph Didymella faba G.J. Jellis & Punith) in fava bean (Vicia faba © XXXX American Chemical Society and American Society of Pharmacognosy
Received: December 7, 2017
A
DOI: 10.1021/acs.jnatprod.7b01034 J. Nat. Prod. XXXX, XXX, XXX−XXX
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The organic extract obtained from the culture filtrates of A. lentis var. lathyri was purified as detailed in the Experimental Section. Five pure metabolites (1−5) were isolated and identified. From a preliminary 1H NMR investigation, two new phytotoxins, named lathyroxins A and B (1 and 2, respectively), appeared to be derivatives of phenol. The other three proved to be well-known metabolites, p-hydroxybenzaldehyde, p-mehoxyphenol, and tyrosol (3−5, respectively). They were identified by comparing their spectroscopic data with those reported in the literature [Ren-Yi et al. (2015)8 for 3, Liu and Ackerman (2013)9 for 4, and Kimura and Tamura (1973),10 Capasso et al. (1992),11 and Cimmino et al. (2017)12 for 5].
being ortho-located to the hydroxy phenol group. The same spectrum showed signals of an ABXY system appearing as a doublet (J = 5.7), a multiplet, and two double doublets (J = 14.1 and 3.7, 14.1, and 8.5 Hz) at δ 4.17, 3.83, 2.92, and 2.67, respectively, and assigned to H-1, H-2, and H-3A and H-3B. The chemical shift of H-1 is typical of a dioxolanemethine, attached to two methoxy groups resonating as singlets at δ 3.48 and 3.46, respectively. As expected, the 1H NMR spectrum also showed two broad singlets due to two hydroxy groups at δ 4.78 and 2.10.14 The couplings observed in the HSQC spectrum15 permitted the assignment of the signals observed in the 13C NMR spectrum (Table 1) at δ 106.0, 72.1, 37.2, 130.6, 115.2, and 54.8 (overlapped signal) to the protonated carbons C-1, C-2, C-3, C-2′,6′, and C-3′,5′ and to the two methoxy groups, respectively.16 The couplings observed in the HMBC spectrum15 (Table 1) between the tertiary oxygenated sp2 carbon (C-4′) and H-2′,6′ and H-3′,5′ identified the carbon at δ 154.1. Similarly the coupling observed between the quaternary sp2 carbon (C-1′) and the same protons (H-2′,6′ and H-3′,5′) and also with H-2 and H2-3 identified the carbon at δ 130.2. Thus, the chemical shifts were assigned to all the protons and the corresponding carbons of 1 as reported in Table 1, and lathyroxin A (1) was formulated as 4-(2-hydroxy3,3-dimethoxypropyl)phenol. The structure assigned to 1 was confirmed by all the couplings observed in the HMBC spectrum (Table 1) and the data from the HRESIMS spectrum. The latter showed the sodiated dimer [2M + Na]+ and the sodium cluster [M + Na]+ at m/z 447 and 235.0931, respectively. Lathyroxin B (2) has a molecular formula of C9H12O3 as deduced by its HRESIMS and consistent with 4 indices of hydrogen deficiency. 2 is related to 1 and differs only by the side chain attached to C-1′, which is a 2,3-diolpropyl instead of 2-hydroxy-3,3-dimethoxypropyl. Indeed, the comparison of their 1H and 13C NMR data (Table 1) showed that the shift and the multiplicity changes of the signal of C-1, being a hydroxymethyl in 2 and a dimethoxymethine in 1, are the only substantial differences between the two compounds. The 1H NMR spectrum of 2 showed two double doublets (J = 11.2 and 4.4, 11.2, and 6.3 Hz, respectively) typical of a hydroxymethyl
Lathyroxin A (1) has the molecular formula C11H16O4 as deduced by its HRESIMS and consistent with 4 indices of hydrogen deficiency. Its 1H and 13C data showed signal systems similar to those of a substituted derivative of phenol. These data are also consistent with the bands of hydroxy and aromatic groups observed in the IR spectrum,13 as well as with the absorption maxima observed in the UV spectrum.14 The 1H (Table 1) and COSY15 spectra showed the typical pattern system of a para-disubstituted benzene ring with two doublets (J = 8.3 Hz) at δ 7.15 (H-2′,6′) and 6.79 (H-3′,5′), the latter
Table 1. 1H and 13C NMR Data of Lathirixins A and B (1 and 2, Respectively) 1 position
δ Cc
2
δH (J in Hz)
δCc
HMBC
δH (J in Hz)
1
106.0 d
4.17 (1H) d (5.7)
H2-3, H-2, OMe
65.1 t
2 3
72.1 d 37.2 t
3.83 (1H) m 2.92 (1H) dd (14.1, 3.7) 2.67 (1H) dd (14.1, 8.5)
H2-3, OMe H-2′, 6′, H-1
73.3 d 38.7 t
1′ 2′, 6′ 3′, 5′ 4′ OMe OMe OH OH OH
130.2 s 130.6 d 115.2 d 154.1 s 54.8 q 54.8 q
H-3′, H-3′, H-2′, H-2′,
129.4 129.9 114.6 155.4
7.15 (2H) d (8.3) 6.79 (2H) d (8.3)
5′, H-2′, 6′, H2-3, H-2 5′, H2-3, H-2 6′ 6′, H-3′, 5′,
s d d s
3.50 3.43 3.76 2.73 2.61
(1H) (1H) (1H) (1H) (1H)
dd dd m dd dd
(11.2, 4.4) (11.2, 6.3) (13.8, 5.9) (13.8, 7.4)
7.06 (2H) d (8.4) 6.72 (2H) d (8.4)
HMBC H2-3, H-2 H-2′, 6′, H2-1, H2-3 H-2′, 6′, H-2 H-3′, 5′, H-2, H2-3 H2-3 H-2′, 6′ H-2′, 6′, H-3′, 5′
3.48 (3H) s 3.46 (3H) s 0.91 (1H) br s 1.31 (1H) br s 3.46 (1H) br s
2.10 (1H) br s 4.78 (1H) br s
a The chemical shifts are in δ values (ppm) from TMS. b2D 1H,1H (COSY) and 13C,1H (HSQC) NMR experiments delineated the correlations of all the protons and the corresponding carbons. cMultiplicities were assigned by DEPT spectrum.
B
DOI: 10.1021/acs.jnatprod.7b01034 J. Nat. Prod. XXXX, XXX, XXX−XXX
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group at δ 3.50 and 3.43,14 which in the HSQC spectrum also coupled with the carbon (C-1) at δ 65.1.16 As expected, the 1H NMR spectrum also showed broad singlets due to three hydroxy groups at δ 3.46, 1.31, and 0.91, respectively. Conversely, the signals present in the 1H and 13C NMR spectra of 1 for the dioxolanemethine and the two methoxy groups were absent in the spectra of 2. The couplings observed in the HSQC and HMBC spectra of 2 (Table 1) supported the assignment of chemical shifts of all the protons and the corresponding carbons of 2 as reported in Table 1 and to formulate lathyroxin B (2) as 3-(4-hydroxyphenyl)propane-1,2diol. The structure assigned to 2 was also supported by the data of its HRESIMS spectrum, which showed the sodiated dimer [2M + Na]+ and the sodium cluster [M + Na]+ at m/z 359 and 191.0663, respectively. Furthermore, the significant tropylium ion [HO−Ph−CH2]+ was recorded at m/z 107, probably generated from the pseudomolecular ion by loss of the 1,2diolpropyl residue. The absolute configuration at C-2 of the side chain of both 1 and 2 was assigned by recording their electronic circular dichroism (ECD) spectra, which were compared to that of the structurally related crypticin B (6) having the same chromophore. 6 was recently isolated from the culture filtrates of Diaporthella cryptica B.T. Linaldeddu, A. Deidda & B. Scanu, the causal agent of halzenut trunk and branch dieback in Sardinia, Italy, and its C-2 S configuration was assigned by experimental and computed ECD spectra.17 The comparison of its spectrum with those of lathyroxins A and B (1 and 2) supported the assignment of an R absolute configuration for C2 of their side chains. 3-(4-Hydroxyphenyl)propan-1,2-diol was previously reported as a fungal18,19 and a plant20 metabolite, but the absolute stereochemistry and biological activities were not. The racemic 3-(4-hydroxyphenyl)propan-1,2-diol has been synthesized starting from 4-chlorophenol and 2-propenol.21 The Senantiomer of lathyroxin B (2) was prepared from acid hydrolysis of the (S)-3-[4-(benzyloxy)phenyl]propane-1,2diol.22 This diol was, in turn, obtained from the reduction of optically pure (S)-3-p-benzyloxyphenyllactic acid synthesized starting from O-benzyl-L-tyrosine.23 Thus, this is the first report of 2 as a natural product. Lathyroxin B (2) caused clear necrosis on all the tested plants. The clearest effects were visible on Sonchus oleraceus L., Lycopersicon esculentum (L.) Karsten ex Farw., Phaseolus vulgaris L., and Lens culinaris Medik. Lathyroxin A (1) was active only on Lupinis albus L. and S. oleraceus, whereas p-hydroxybenzaldehyde was active only on L. albus and L. culinaris. The other two compounds were inactive at the tested concentrations. On seeds of the parasitic weed Phelipanche ramosa, lathyroxin B (2) reduced germination by 63% (data reported in the SI). Lathyroxin A (1) caused approximatively 36% inhibition. Both compounds clearly shortened the germination tubes of the seeds. On Lepidium sativum, lathyroxin B (2) reduced the rootlet length of the germinated seeds by over 50% (data reported in the SI). Lathyroxin A (1) and tyrosol showed a modest but still significant reduction of rootlet elongation by around 15% (data reported in Table S1), whereas the other two compounds were inactive. In the other assays against arthropods, nematodes, and bacteria (described in detail in the SI) all the compounds were inactive.
In conclusion two monosubstituted phytotoxic phenol derivatives were isolated from the culture filtrates of A. lentis var. lathyri and named lathyroxins A and B (1 and 2). 1 is a new natural compound, while 2 is reported for the first time as a phytotoxic metabolite produced by the same fungus. pHydroxybenzaldehyde (3) is a natural product produced by several plants, insects, and fungi.24 As a phytotoxin it was isolated from Botryosphaeria obtusa (Schwein.) Shoemaker,24 Phaeoacremonium aleophilum W. Gams, Crous, M.J. Wingf. & Mugnai, Fomitiporia mediterranea M. Fisch.,25 and Diaporthe gulyae R.G. Shivas, S.M. Thompson & A.J. Young.26 Its inhibitory effect on seed germination was determined on wheat (Triticum aestivum L.) and radish (Raphanus sativus L.).8 Recently 3 was also isolated from the Fusarium strain belonging to the F. tricinctum species complex showing phytotoxicity against Bromus tectorum L.27 p-Methoxyphenol (4) was isolated from the culture broth of Colletotrichum aotearoa BCRC 09F0161, an endophytic fungus that infected leaves of an endemic Formosan plant, Bredia oldhamii Hook. f. (Melastomataceae),28 and from the twigs of Dorstenia turbinate Engl.29 Tyrosol (5) is a well-known fungal metabolite produced by several pathogens, including Diplodia seriata De Not. and Neof usicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips.30−35 Its phytotoxic activity on leaves of marigold (Tagetes erecta L.), pricklysida (Sida spinosa L.), and lamb’s quarters (Chenopodium album L.) was previously reported.30,33 Tyrosol was also recently reported from the halzenut pathogen Diporthella cryptica isolated in Sardinia, Italy.17
Figure 1. ECD spectra of lathyroxin A (pink line), lathyroxin B (green line), and crypticin B (brown line).
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EXPERIMENTAL SECTION
General Experimental Procedures. IR spectra were recorded as deposited on a glass film on a Thermo Nicolet 5700 FT-IR spectrometer; UV spectra were measured in MeOH on a Jasco V530 spectrophotometer; CD spectra were recorded in MeCN on a Jasco J-815 circular dichroism spectropolarimeter, calibrated for intensity with ammonium [D10] camphorsulfonate ([θ]290.5 = 7910 deg cm2 dmol−1). The Hellma cells were made of quartz Suprasil, and the path length was 0.2 cm. 1H, 13C, and 2D NMR spectra were recorded at 400 or 500 MHz in CDCl3 or CD3OD on Bruker and Varian instruments. The same solvents were also used as internal standards. Carbon multiplicities were determined by DEPT spectra.15 DEPT, COSY-45, HSQC, HMBC, and NOESY experiments were performed using Bruker and Varian microprograms.15 HRESI and ESI spectra were recorded on a Waters Q-TOF Micro Mass and on an Agilent 6120 Quadrupole LC/MS instrument, respectively. Analytical, preparative, and reverse-phase TLCs were carried out on silica gel C
DOI: 10.1021/acs.jnatprod.7b01034 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Leaf Puncture Assay. The five metabolites mentioned above were tested at 2 μg/μL concentration on seven different plant species, belonging to five botanical families, i.e., Lupinus albus, Lens culinaris, Phaseolus vulgaris (Fabaceae family), Sonchus oleraceus (Asteraceae), Convolvulus arvensis L. (Convolvulaceae), Lycopersicon esculentum (Solanaceae), and Mercurialis annua L. (Euphorbiaceae). Droplets (20 μL) of the solution containing the compound were applied to detached leaves previously punctured with a needle. Five replications were used for each plant species tested. Leaves were kept in a moistened chamber under continuous fluorescent light at 25 °C. The eventual appearance of symptoms, consisting of circular necrosis, was observed 3 days after droplet application. Control treatments were carried out by applying droplets of a methanol solution (1% MeOH). Assay on Seed Germination of the Parasitic Weed Phelipanche ramosa. The assay was carried out by following an experimental scheme previously described.36 Due to the novelty of the compounds, in this study only lathyroxins A and B were tested on conditioned P. ramosa seeds at 10−3 and 10−4 M concentrations, by contemporaneous application of the synthetic stimulant GR24. After 3 days, the number of germinated seeds was determined by direct observation under a stereomicroscope, and the results were expressed as germination reduction percentage in comparison to the control. Visual estimation of the rootlet length was also carried out. The experiment was carried out in duplicate. Assay on Lepidium sativum Rootlet Elongation. Seeds of L. sativus were rinsed for several minutes under a continuous flux of tap water. Thus, they were germinated by placing them on Petri dishes, on a wet paper disk, at 25 °C in the dark for 24 h. Twenty uniformly germinated and healthy seedling were selected and transferred to each smaller Petri dish, containing the paper disk wetted with the test solution. After 3 days the rootlet length was measured, and the results were expressed as percentage reduction of rootlet length.
(Merck, Kieselgel 60, F254, 0.25, 0.5 mm and RP-18 F254s, respectively) plates. The spots were visualized by exposure to UV radiation or by spraying first with 10% H2SO4 in MeOH and then with 5% phosphomolybdic acid in EtOH, followed by heating at 110 °C for 10 min. Column chromatography was performed using silica gel (Merck, Kieselgel 60, 0.063−0.200 mm). Fungal Strain. The strain of Ascochyta lentis var. lathyri used in this study was isolated from a diseased Lathyrus sativus (grass pea) plant in Italy3 and stored in the fungal collection of the Istituto di Scienze delle Produzioni Alimentari, CNR, Italy, with the code ITEM 17453. The isolate was routinely grown and maintained in plates and slants containing potato-dextrose agar (PDA, Sigma−Aldrich, Chemie Gmbh, Buchs Switzerland). Production, Extraction, and Purification of Ascochyta lentis var. lathyri Secondary Metabolites. The fungus was grown in 1 L Roux flasks containing 200 mL of a defined mineral for 4 weeks at 25 °C in the dark. The culture filtrate (7 L), obtained by filtration, was lyophilized and stored at −20 °C until use. It was redissolved in distilled water at 1/10 of its initial volume, acidified to pH 2 with HCOOH, and extracted exhaustively with EtOAc. The combined organic extracts were dried with Na2SO4 and evaporated under reduced pressure. The residue (2.3 g) was fractionated by column chromatography on silica gel eluted with CHCl3−i-PrOH (9:1), and eight groups of homogeneous fractions were obtained. The residue (64.1 mg) of the fourth fraction was further purified by preparative TLC eluted with CHCl3−MeOH (95:5), obtaining an amorphous homogeneous solid directly identified as below reported as phydroxybenzaldehyde (3, 1.9 mg, 0.3 mg/L, Rf 0.5). The residue (313.5 mg) of the fifth fraction was purified by column chromatography on silica gel eluted with CH2Cl2−MeOH (9:1), giving 11 homogeneous fractions. The residue (14.1 mg) of the fourth fraction was further purified by two successive TLC steps, using Me2CO−H2O (1:1) and n-hexane−Me2CO (6:4), respectively, yielding an amorphous solid, named lathyroxin A (1, 4.8 mg, 0.7 mg/L, Rf 0.7). The residue (14.1 mg) of the fifth fraction was purified by reverse-phase TLC, eluted with Me2CO−H2O (1:1), allowing to obtain an amorphous solid identified, as below reported, as tyrosol (5, 3.2 mg, 0.5 mg/L, Rf 0.6). The residue (33.0 mg) of the sixth fraction of the same column, purified by two successive TLC steps using nhexane−Me2CO (1:1) and Me2CO−H2O (4:6), respectively, yielded an amorphous solid named lathyroxin B (2, 20.1 mg, 2.9 mg/L, Rf 0.7). Lastly, the residue (273.4 mg) of the seventh fraction of the initial column was purified by column chromatography on silica gel eluted with CHCl3−i-PrOH (85:15), giving seven fractions. The residue (43.3 mg) of the fourth fraction was purified by reverse-phase TLC using MeCN−H2O (4:6), yielding an amorphous solid identified as pmethoxyphenol (4, 2.7 mg, 0.4 mg/L, Rf 0.7). Lathyroxin A (1): UV λmax (log ε) 278 (3.17), 223 (3.65) nm; IR νmax 3358, 1613, 1516, 1549 cm−1; 1H and 13C NMR, see Table 1; HRESIMS (+) m/z 447 [2M + Na]+ and 235.0931 [M + Na]+ (calcd for C11H16O4Na 235.0946). Lathyroxin B (2): UV λmax (log ε) 278 (3.07), 224 (356) nm; IR νmax 3364, 1613, 1569, 1458 cm−1; 1H and 13C NMR, see Table 1; HRESIMS (+) m/z 359 [2M + Na]+, 191.0663 [M + Na]+ (calcd for C9H12O3Na 191.0684) and 107 [HO−Ph−CH2]+. p-Hydroxybenzaldehyde (3): 1H NMR δ 9.90 (s, CHO), 7.83 (d, J = 7.9 Hz, H-2 and H-6), 6.98 (d, J = 7.9 Hz, H-3 and H-5), 4.70 (s, OH). These data are in agreement with those previously reported;8 ESIMS (+) m/z 145 [M + Na]+. p-Methoxyphenol (4): 1H NMR (500 MHz, in CDCl3) δ 7.05 (d, J = 8.5 Hz, H-3 and H-5), 6.80 (d, J = 8.5 Hz, H-2 and H-6), 4.69 (s, OH), 3.64 (s, OCH3). These data are in agreement with those previously reported;9 HRESIMS (+) m/z 269.07452 [2M + Na] + (calcd for C14H14O4Na+, 269.07843) and 107.04869 [M − H2O]+ (calcd for C7H7O+, 107.04969). Tyrosol (5): 1H NMR (500 MHz, in CDCl3) δ 7.12 (d, J = 6.5 Hz, H-2 and H-6), 6.81 (d, J = 6.5 Hz, H-3 and H-5), 4.87 (s, OH), 3.85 (t, J = 6.5 Hz, H-8 and OH), 2.82 (t, J = 6.5 Hz, H-7); ESIMS (+) m/z 299 [2M + Na]+, 161 [M + Na]+. These data are in agreement with those previously reported.10−12
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b01034. NMR, HRESIMS, IR, and UV spectra of compounds 1 and 2 and other details on biological activities (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel: +39 081 2539178. Fax: +39 081 674330. E-mail:
[email protected]. ORCID
Alessio Cimmino: 0000-0002-1551-4237 Antonio Evidente: 0000-0001-9110-1656 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The work was carried out with academic grants from the Dipartimento Scienze Chimiche, Università di Napoli Federico II, Napoli, Italy. A.E. is associated with Istituto di Chimica Biomolecolare del CNR, Pozzuoli, Italy. Prof. A. Lombardi and Dr. L. Lista are thanked for help with ECD measurement. The authors thank T. D’Addabbo and F. Catalano, CNR-ISPP, for the technical assistance with the nematode bioassay, and N. Montemurro, CTER ISPA, for technical assistance.
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REFERENCES
(1) Vaz-Patto, M. C.; Rubiales, D. Ann. Bot. 2014, 113, 895−908.
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DOI: 10.1021/acs.jnatprod.7b01034 J. Nat. Prod. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.jnatprod.7b01034 J. Nat. Prod. XXXX, XXX, XXX−XXX
