Cyclobotryoxide, a Phytotoxic Metabolite Produced by the Plurivorous

Article pubs.acs.org/jnp

Cyclobotryoxide, a Phytotoxic Metabolite Produced by the Plurivorous Pathogen Neof usicoccum australe Anna Andolfi,† Lucia Maddau,‡ Alessio Cimmino,† Benedetto T. Linaldeddu,‡ Antonio Franceschini,‡ Salvatorica Serra,‡ Sara Basso,† Dominique Melck,§ and Antonio Evidente*,† †

Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali, Università di Napoli Federico II, Via Università 100, 80055 Portici, Italy ‡ Dipartimento di Agraria, Sezione di Patologia Vegetale ed Entomologia, Università degli Studi di Sassari, Via E. De Nicola 1, 07100 Sassari, Italy § Istituto di Chimica Biomolecolare del CNR, Comprensorio Olivetti, Edificio 70, Via Campi Flegrei 34, 80078 Pozzuoli, Italy ABSTRACT: Two isolates of Neof usicoccum australe belonging to ITS haplotypes H4 and H1 and associated with grapevine cordon dieback and branch dieback of Phoenicean juniper, respectively, have been shown to produce in vitro structurally different secondary metabolites. From the strain BOT48 of N. australe (haplotype H4) a new cyclohexenone oxide, namely, cyclobotryoxide, was isolated together with 3-methylcatechol and tyrosol. Cyclobotryoxide was characterized as (1S,5R,6S)-5-hydroxy-3-methoxy-4-methyl-7-oxabicyclo[4.1.0]hept-3-en-2-one by spectroscopic, optical, and chemical methods. The strain BL24 (haplotype H1) produced tyrosol along with botryosphaerone D and (3S,4S)-3,4,8-trihydroxy-6-methoxy-3,4dihydro-1(2H)-naphthalenone. The metabolites obtained from both strains were tested at four concentrations on leaves of grapevine cv. Cannonau, holm oak, and cork oak by the leaf puncture assay. Cyclobotryoxide proved to be the most phytotoxic compound. Tyrosol and cyclobotryoxide were also tested on detached grapevine leaves at concentrations of 0.25 and 0.5 mg/mL. Only cyclobotryoxide was found to be active in this bioassay.

T

significant variations in virulence among the strains of this plurivorous pathogen. The nature of symptoms caused by N. australe on grapevine and Phoenicenan juniper (Figure 1A and B) suggests that phytotoxic metabolites may be involved in the host−pathogen interaction. On the other hand, it is well-known that Botryosphaeriaceae species are able to produce in vitro a plethora of bioactive secondary metabolites, some of which may contribute to the onset of disease symptoms.11−15 In particular, Botryosphaeriaceae species associated with declining grapevine are able to produce hydrophilic and lipophilic bioactive metabolites in liquid culture.16 So far, phytotoxins have been isolated from Neof usicoccum parvum17 and Diplodia seriata,18 although their role on symptom disease expression needs to be fully elucidated. While new polyketides along with other known secondary metabolites were isolated from one isolate of N. australe belonging to haplotype H12,7 no data on the production of phytotoxic metabolites by others N. australe haplotypes have been published. Given that significant variation in the virulence between the isolates was observed, it is reasonable to assume that some variation on the secondary metabolite profiling could occur among isolates of this species. Thus, the objective of this research was to isolate and characterize the phytotoxic metabolites produced in vitro by two strains of N. australe belonging to H1 and H4 haplotypes isolated from Phoenicean juniper and grapevine, respectively.

he plurivorous pathogen Neof usicoccum australe (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips, originally thought to be native to the southern hemisphere,1,2 has recently been reported on a variety of native and exotic hosts worldwide.3−7 In Italy N. australe was primarily found associated with drupe rot of olives8 and declining grapevine plants.9 Phylogenetically a one bp difference in the sequence of the ITS region of rDNA was observed between the Italian isolates of N. australe and the ex-type isolate (CMW6837) used in the original description.8,9 Recently Sakalidis et al.,10 reevaluating GenBank ITS rDNA sequence data of N. australe, found the occurrence of 13 ITS haplotypes for this fungal species, of which two were dominant (H1 and H4), while the others were more rare. On the basis of the findings of this extensive study, Italian strains of N. australe fit within the haplotype H4. However, in a study still in progress aimed at clarifying the causes of decline affecting Juniperus spp. in several natural areas in Sardinia (Italy), a large collection of N. australe strains were isolated from juniper trees showing a progressive dieback of shoots and branches. Phylogenetically most of the juniper’s strains of N. australe fit mainly within the haplotype H4, but some strains belong to the haplotype H1. These findings suggest that N. australe is part of the endemic mycobiota of native tree species in Sardinia, in addition to being a pathogen of agricultural crops. Pathogenicity tests conducted on grapevine and juniper plants using Sardinian strains of N. australe belonging to both H4 and H1 haplotypes have revealed the occurrence of © 2012 American Chemical Society and American Society of Pharmacognosy

Received: July 23, 2012 Published: October 9, 2012 1785

dx.doi.org/10.1021/np300512m | J. Nat. Prod. 2012, 75, 1785−1791

Journal of Natural Products

Article

Figure 2. Structures of cyclobotryoxide and its acetyl derivative, 3methylcatechol, tyrosol, botryosphaerone D, and (3S,4S)-3,4,8trihydroxy-6-methoxy-3,4-dihydro-1(2H)-naphthalenone (1−6) produced by N. australe from grapevine and juniper.

corresponding to a molecular formula of C8H10O4, indicating four hydrogen deficiencies. The same spectrum showed the sodium cluster [M + Na]+ at m/z 193.1509, and when the same spectrum was recorded in negative mode, the pseudomolecular ion [M − H]− at m/z 169 appeared. The UV spectrum showed an absorption maximum at 275 nm, consistent with the theoretical one (272) calculated applying the Woodward rules for compound containing a suitable α,β,γ-trisubstituted α,βunsaturated carbonyl group.19 The IR spectrum showed bands characteristic of hydroxy and conjugated carbonyl groups.20 The 1H NMR spectrum of 1 (Table 1) showed the presence of a doublet of doublets (J = 3.0 and 1.0 Hz) and a doublet (J = Table 1. 1H and 13C NMR Data and HMBC Correlations for Cyclobotryoxide (1) in CDCl3a,b δCc

δH

HMBC

1 2 3

52.1 (d) 194.3 (s) 165.5 (s)

3.45 (1H, d, J = 3.0 Hz)

4 5 6

112.0 (s) 60.9 (d) 54.2 (d)

H-5, H-6 H-1, CH3 CH3, OH, H-1, OCH3, H-5 H-5, OH, CH3 H-6, OH, CH3 H-5, H-1

position

Figure 1. Grapevine and juniper diseases symptoms associated with Neof usicoccum australe strain BOT48 (A) and BL24 (B), respectively.

■

OCH3 CH3 OHd

RESULTS AND DISCUSSION The two strains of N. australe, belonging to haplotypes H4 and H1, were fermented in liquid Cazapek medium containing 2% corn meal. The cultures were exhaustively extracted with EtOAc, and the organic extracts were purified by combined column and TLC chromatography as detailed in the Experimental Section, yielding five metabolites (1, 3−6, Figure 2). Metabolites 1, 3, and 4 were produced by haplotype H4, while metabolites 4, 5, and 6 were identified for haplotype H1. All the metabolites were isolated as homogeneous amorphous solids and by preliminary 1H NMR investigation appeared to belong to different classes of natural compounds. The compound named cyclobotryoxide (1) had a molecular weight of 170 as deduced from its HRESIMS spectrum,

56.1 (q) 7.9 (q)

4.88 (1H, 3.79 (1H, Hz) 4.05 (3H, 1.26 (3H, 3.60 (1H,

multiplet) dd, J = 3.0, 1.0 s) br s) d, J = 11.0 Hz)

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. c Multiplicities were assigned by DEPT spectrum. dThe shift is variable; this value was observed in CDCl3 at room temperature. a

3.0 Hz) at δ 3.79 and 3.45, respectively, typical of the protons (H-6 and H-1) of a cis-disubstituted oxirane ring.19 The resonance at δ 4.88 was assigned to a proton (H-5) of a secondary hydroxylated carbon. It coupled in the COSY spectrum21 with the geminal hydroxy group observed as a doublet (J = 11.0 Hz) at δ 3.60, with H-6 and long-range (J < 1 1786



Article

Hz) with a vinyl methyl group [CH3−C(4)]. The latter was observed as a broad singlet at δ 1.26. Furthermore, the 1H NMR spectrum showed a singlet at δ 4.05 assigned to a methoxy group.19 These partial structures were confirmed by data of the 13C NMR spectrum (Table 1), which showed the presences of signals due to an α,β-unsaturated α-oxygenated carbonyl group (δ 194.3, 165.5, 112.0), for the carbonyl (C-2) and two quaternary olefinic (C-3 and C-4) carbons. The methoxy and methyl groups resonated at δ 56.1 and 7.9 and were located at C-3 and C-4, respectively, on the basis of the couplings observed in the HMBC spectrum.21 The three oxygenated secondary carbons were observed at typical chemical shift values of δ 60.9, 54.2, and 52.1, and they were assigned by the coupling observed in the HSQC spectrum21 to C-5, C-6, and C-1, respectively.22 Thus, chemical shifts were assigned to all the protons and carbons, and cyclobotryoxide was formulated as 5-hydroxy-3methoxy-4-methyl-7-oxabicyclo[4.1.0]hept-3-en-2-one. This structure was confirmed by all the couplings and correlations observed in the HMBC and NOESY21 spectra. The NOESY spectrum showed, besides the expected correlations between H-1 and H-6 and the methoxy and the methyl groups, also those between the latter and the proton of the hydroxy group at C-5 and between H-5 and H-6. The latter correlation was in agreement with an inspection of a Dreiding model of 1, as both relative configurations of C-5 (R* or S*) could generate an NOE effect between H-6 and H-5. However, only the relative R* configuration could be assigned to C-5, as it is consistent with the dihedral angle near 90° observed between H-5 and H-6, which, in agreement with the literature, had a very low value (1.0 Hz) measured for their coupling in the 1H NMR spectrum.19 The absolute configuration of 1 was determined by comparison of its ECD spectrum with those of the structurally related epoxycyclohexenones.23 In fact, its ECD spectrum (Figure 3A) showed negative and positive Cotton effects at 310 and 276 nm, respectively. These effects were similar to related epoxycyclohexenones such as sphaeropsidone, terremutin, and panepoxydon.11,23 On the basis of these results 1 was formulated as (1S,5R,6S)5-hydroxy-3-methoxy-4-methyl-7-oxabicyclo[4.1.0]hept-3-en-2one. The structure assigned to cyclobotryoxide was further supported by preparation of derivative 2 by acetylation. The spectroscopic data of this derivative were fully consistent with the structure assigned to 1. The IR spectrum of 2 showed the absence of a hydroxy group and the presence of a band typical of an acetyl carbonyl group at 1747 cm−1.20 Its 1H NMR spectrum differed from that of 1 essentially in the downfield shift (Δ δ 1.30) of H-5 observed as a broad singlet at δ 6.18 and the absence of the doublet at δ 3.60 of the hydroxy proton and the presence of the singlet of the acetyl group at δ 2.19. The ESIMS spectrum of 2 recorded in the positive mode showed the sodium cluster [M + Na]+ at m/z 235. Metabolite 3 showed a molecular weight of 124, as deduced from its ESIMS spectrum recorded in the positive and negative modes, and was identified as 3-methylcatechol on the basis of its spectroscopic data, which were the same as previously reported.24 3-Methylcatechol, a minor metabolite of toluene, showed toxicity in human and mammal cells.25−27 It was also isolated, together with other phenolic compounds, from the

Figure 3. ECD spectra of cyclobotryoxide (A) and botryosphaerone D (B) recorded in EtOH.

fermentation liquid of the endophytic fungus Penicillium sp. GT6105 from Kandelia candel.28 Metabolite 4 was isolated from the organic extracts of both strains of N. australe and was identified as tyrosol, on the basis of its 1H NMR spectrum, which was similar to that previously reported.29 The ESIMS spectrum showed the sodium cluster [M + Na]+ and the pseudomolecular ion [M − H]− at m/z 161 and 137, respectively, when it was recorded in the positive and negative modes. Tyrosol is a phytotoxic metabolite produced by plants and fungi including Botryosphaeriaceae species.15,29 Recently, tyrosol was also identified as a phytotoxic metabolite produced by N. parvum.17 This fungus was isolated together with four other Botryosphaeriaceae from declining grapevines in Spain.16 From the organic extract of N. australe isolated from juniper, metabolites 5 and 6 were obtained. The molecular weights of 5 and 6 were determined to be 252 and 224, respectively, as deduced from their ESI and APCIMS spectra recorded, respectively, in the positive and negative modes. The chemical shifts and multiplicities of all protons and corresponding carbons of 5 and 6 were assigned by the couplings observed in their COSY, HMBC, and HSQC spectra. Comparison of these and reported data identified 5 and 6 as botryosphaerone D7 and (3S,4S)-3,4,8-trihydroxy-6-methoxy3,4-dihydro-1(2H)-naphthalenone.30 The absolute configuration of botryosphaerone D (5), not previously reported, was also determined. Its ECD spectrum (Figure 3B) was recorded under the same conditions and compared to that of 6.30 On the basis of coincident negative and positive Cotton effects at 214, and 239 and 280 nm, 1787



Article

Table 2. Phytotoxicity of Compounds 1−6 Evaluated at Different Concentrations on Three Plant Species [Area Lesions (mm2) ± SE] concentration (mg/mL) compounds

species

1

cork oak holm oak grapevine cork oak holm oak grapevine cork oak holm oak grapevine cork oak holm oak grapevine cork oak holm oak grapevine cork oak holm oak grapevine

2

3

4

5

6

a

1 53.1 27.7 24.3 13.1 19.4 6.9 1.0 1.0 4.9 0.9 0.8 8.3 4.8 3.3 11.9 n.a.a n.a. n.a.

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.5 1.7 4.9 1.1 1.6 2.8 0.4 0.1 0.1 0.9 0.2 0.05 1.3 1.6 0.6 2.3

22.4 5.5 5.2 5.2 3.5 3.4 n.a. n.a. 1.9 n.a. n.a. n.a. 2.3 n.a. 2.5 n.a. n.a. n.a.

± ± ± ± ± ±

4.3 1.9 0.3 1.7 1.9 0.4

± 0.5

± 0.5 ± 0.3

0.25

0.125

± ± ± ± ± ±

n.a. n.a. 0.9 ± 0.06 n.a. n.a. 0.8 ± 0.06 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.

4.5 1.9 1.2 2.4 1.4 1.3 n.a. n.a. 1.0 n.a. n.a. n.a. 0.8 n.a. 0.6 n.a. n.a. n.a.

1.3 0.7 0.2 0.9 0.4 0.2

± 0.2

± 0.3 ± 0.1

n.a. = no activity.

showed moderate activity on grape leaves, while on the other species tested it was weakly active or inactive. Compound 4, produced by both N. australe isolates, exhibited moderate phytotoxicity on grapevine leaves, whereas on the other species tested it was weakly active or inactive. Compound 5, isolated as the main metabolite of haplotype H4, appeared to be much less toxic than 1 in this bioassay, even at the highest concentration. In addition, compound 6 did not show any phytotoxicity either. Compounds 1 and 4 were also tested on grapevine detached leaves at 0.25 and 0.5 mg/mL. In this bioassay cyclobotryoxide caused symptoms at 0.5 mg/mL on the foliar lamina consisting of irregular internerval or marginal necrotic spots, slowly becoming coalescent, followed by distortion and withering of the leaf lamina. No visible symptoms were induced by tyrosol. The present study provides the first report on the expression of phytotoxic secondary metabolites by two N. australe isolates belonging to two of the 13 haplotypes of this species proposed by Sakadilis et al.10 The metabolites characterized emphasized different metabolic profiles of the two N. australe haplotypes (H1 and H4) examined. Such diversity is also reflected in the toxic potential shown by each of the metabolites isolated. This is not surprising, considering that differences in aggressiveness within N. australe isolates correlate with genetic variability as previously reported.34,35 A different degree of virulence has been observed for fungal pathogens, and in some cases these differences were correlated with their different capacity to produce virulence factors involved, fully or in part, in the symptom expression.36−38 An aspect worthy of discussion concerns the nature of the symptoms induced by N. australe on grapevine. To date, field observations have shown that this pathogen is chiefly involved in the etiology of canker and dieback of grapevine trunk and cordon. Therefore, it is interesting to understand if the phytotoxins secreted by this pathogen could also be involved in the onset of foliar symptoms on the host plant as already reported for other grapevine pathogens. It is likely that the symptomatology associated with infection of N. australe on grapevine has not yet been clarified. In this regard, our research

compound 5 was formulated as (3S,4S)-7-ethyl-3,4,8-trihydroxy-6-methoxy-3,4-dihydro-1(2H)-naphthalenone. Botryosphaerone D (5), together with three other new naphthalenone polyketides, had previously been isolated from N. australe strain ZJ12-1A, a fungus obtained from sterilized root epidermis of Sonneratia apetala in China. This compound did not show any antimicrobial and cytotoxic activities.7 Finally, (3S,4S)-3,4,8trihydroxy-6-methoxy-3,4-dihydro-1(2H)-naphthalenone (6) was reported as a new naphthalenone produced from wooddecaying fungus Phaeosphaeria sp.30 Cyclobotryoxide was characterized as a new naturally occurring compound belonging to a family of epoxycyclohexenones that have been isolated from bacteria, fungi, higher plants, and mollusks. These compounds have shown interesting biological properties such as antifungal, antibacterial, antitumoral, phytotoxic, and enzyme-inhibitory activities.23 Furthermore, sphaeropsidone and epi-sphaeropsidone, belonging to the same family of natural compounds, were previously isolated from Diplodia cupressi,31 a pathogenic fungus of cypress. The isolation of naphthalenone polyketides as 5 and 6 was not a surprise. In fact, they belong to a group of well-known phytotoxins produced by pathogenic fungi for different agrarian crops such as scytalone, isosclerone, botrytone, and cis- and trans-3,4dihydro-2,4,8-trihydroxynaphthalenones.32,33 When assayed on the leaves of different plant species at different concentrations, compounds 1 and 3−6 showed varying degrees of phytotoxicity. The toxicity data obtained in the leaf-puncture bioassay on holm oak, cork oak, and grapevine cv. Cannonau leaves are summarized in Table 2. Cyclobotryoxide (1) appeared to be the most active metabolite. It had remarkable toxicity in a range of concentrations from 0.25 to 1.0 mg/mL, causing necrotic lesions on leaves of all species tested. At the lower concentrations its toxicity was progressively reduced. A marked decrease of activity was observed for its acetyl derivative (2) even at the highest concentration (1 mg/mL) tested, confirming that structural modifications could alter biological activity. At the lower doses, compound 2 was inactive on cork and holm oak. Compound 3 1788



Article

chromatography on silica gel, eluted with CHCl3/i-PrOH (95:5, v/ v). Eleven homogeneous fraction groups were collected and screened for their phytotoxic activity. The residue (154.1 mg) of the fifth fraction was further purified by CC on silica gel, eluted with petroleum ether/Me2CO (7:3). The residue (38.4 mg) of the fifth fraction was further purified by TLC on silica gel, eluent n-hexane/Me2CO (7:3), yielding a homogeneous, amorphous solid [1, 23.7 mg, Rf 0.53, eluent n-hexane/Me2CO (7:3), Rf 0.50, eluent CHCl3/i-PrOH (95:5)], which was named cyclobotryoxide. The residue (100.7 mg) of the sixth fraction from the initial column was further purified by CC on silica gel, eluted with CHCl3/MeOH (97:3). The residue (40.8 mg) of the third fraction was purified by TLC on silica gel, eluent n-hexane/ Me2CO (7:3), yielding a further quantity of 1 (8.7 mg, total 5.7 mg/ L). The residue (20.1 mg) of the fourth fraction was purified by two successive TLC steps on silica gel, eluted with CHCl3/i-PrOH (95:5) and EtOAc/n-hexane (55:45), yielding a homogeneous, amorphous solid identified, as below reported, as 3-methylcatechol [3, 4.4 mg, 0.8 mg/L, Rf 0.50, eluent CHCl3/i-PrOH (92:8), Rf 0.61, eluent EtOAc/nhexane (55:45)]. The residue (86.9 mg) of the eighth fraction of the initial column was purified by two successive steps using CC and TLC on silica gel, both eluted with CHCl3/i-PrOH (95:5), yielding a homogeneous, amorphous solid that was identified as tyrosol [4, 1.7 mg, 0.31 mg/L, Rf 0.23, eluent CHCl3/i-PrOH (95:5), Rf 0.48, eluent EtOAc/n-hexane (6:4)]. Cyclobotryoxide (1): [α]25D −73 (c 0.3); ECD (c 6.5 × 10−4 M) Δε −74.4 (310), +14,73 (276); UV λmax nm (log ε) 275 (4.02); IR νmax cm−1 3346, 1712, 1640, 1613, 1462, 1243; 1H and 13C NMR, Table 1; HRESIMS (+) spectrum m/z 193.1509 [C8H10O4Na, calcd 193.1518, M + Na]+; ESI (−) m/z 169 [M + H]−. 3-Methylcathecol (3): UV λmax nm (log ε) 292 (3.59); IR νmax cm−1 3318, 1503, 1201; 1H and 13C NMR were similar to those reported;24 ESIMS (+) m/z 147 [M + Na]+; ESIMS (−) m/z 123 [M − H]−. Tyrosol (4): 1H NMR was similar to data reported;29 ESIMS (+) m/z 161 [M + Na]+; ESIMS (−) m/z 137 [M − H]−. Acetylation of Cyclobotryoxide (2). Cyclobotryoxide (1, 5.0 mg), dissolved in pyridine (30 μL), was converted into the corresponding 5-O-acetyl derivative by acetylation with Ac2O (30 μL) at room temperature for 10 min. The reaction was stopped by addition of MeOH, and an azeotrope formed by addition of benzene was evaporated in a N2 steam. The oily residue (6.0 mg) was purified by TLC on silica gel, eluent n-hexane/Me2CO (6:4), yielding derivative 2 as a homogeneous solid (4.1 mg, Rf 0.39). 2: UV λmax nm (log ε) 272 (3.98); IR νmax cm−1 1747, 1654, 1624, 1240, 1207; 1H NMR δ 6.18 (1H, br s, H-5), 3.76 (3H, s, OCH3), 3.63 (1H, dd, J = 1.8 and 1.0 Hz, H-6), 3.50 (1H, d, J = 1.8 Hz, H-1), 2.19 (3H, s, MeCO), 1.76 (3H, br s, Me); ESIMS (+) m/z 235 [M + Na]+. Extraction and Purification of Phytotoxins by N. australe Strain BL24. The culture filtrate (7 L) of this strain, grown in the same cultural conditions of haplotyte H4, was acidified to pH 4 with 2 M HCl and then exhaustively extracted with EtOAc. The organic extracts were combined, dried (Na2SO4), and evaporated under reduced pressure to yield a brown solid residue (400 mg). The residue was tested at concentrations of 4 mg/mL as described below, was found to be phytotoxic against grapevine leaves, and was submitted to bioassay-guided fractionation through column chromatography on silica gel, eluted with CHCl3/i-PrOH (95:5). Ten homogeneous fraction groups were collected and screened for their phytotoxic activity. The residue (11.3 mg) of the sixth fraction was purified by TLC on silica gel, eluent CHCl3/i-PrOH (95:5), yielding a homogeneous, amorphous solid identified as tyrosol [4, 2.8 mg, 0.5 mg/L Rf 0.23, eluent CHCl3/i-PrOH (95:5), Rf 0.48, eluent EtOAc/nhexane (6:4)]. The residue (20.6 mg) of the seventh fraction of the first CC was further purified by two successive steps by TLC on silica gel, eluent CHCl3/i-PrOH (95:5), and by reversed-phase TLC, eluent EtOH/H2O (6:4), yielding two homogeneous, amorphous solids identified as botryosphaerone D [5, mg 4.8, 0.7 mg/L Rf 0.24, eluent CHCl3/i-PrOH (95:5), Rf 0.56, eluent EtOH/H2O (6:4)] and (3S,4S)-3,4,8-trihydroxy-6-methoxy-3,4-dihydro-1(2H)-naphthalenone

group has planned further investigations aimed at clarifying this topic. Furthermore, the current work contributes to expanding the knowledge on bioactive metabolites produced by Botryosphaeriaceae. On the other hand, it is worth pointing out that most of the metabolites so far isolated from Botryosphaeriaceae associated with grapevine trunk disease do not seem to be structurally correlated with those produced by Eutypa lata. This information, previously pointed out by Mahoney et al.,39 could allow Eutypa dieback to be discriminated from other grapevine diseases. In addition, new scenarios of investigation could be opened up for tyrosol, a metabolite frequently isolated from species of Botryosphaeriaceae. Is there a strategy of regulation of virulence factors in Botryosphaeriaceae as also reported for other fungi?40 If so, is tyrosol a quorum-sensing molecule as observed in Candida albicans?41 This is a new challenge for the future.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured in CHCl3, unless otherwise noted, on a Jasco polarimeter, while the ECD spectra were recorded on a JASCO J-815 spectrometer in EtOH; IR spectra were recorded as glassy films on a Perkin-Elmer Spectrum One FT-IR spectrometer, and UV spectra were taken in MeOH solution on a Perkin-Elmer Lambda 25 UV/vis spectrophotometer; 1H and 13C NMR spectra were recorded at 600 and at 125 MHz, respectively, in CDCl3, unless otherwise noted, on Bruker spectrometers. The same solvent was used as internal standard. DEPT, COSY-45, HSQC, HMBC, and NOESY experiments21 were performed using standard Bruker microprograms. HRESIMS spectra were recorded on a Waters Micromass Q-TOF Micro; ESI and APICMS spectra were recorded on an Agilent Technologies 6120 Quadrupole LC/MS instrument. Analytical and preparative TLC were performed on silica gel (Merck, Kieselgel 60, F254, 0.25 and 0.5 mm, respectively) or reversed-phase (Whatman, KC18 F254, 0.20 mm) plates. The spots were visualized by exposure to UV radiation (253) 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 on a silica gel column (Merck, Kieselgel 60, 0.063−0.200 mm). Fungal Strains, Culture Medium, and Growth Conditions. Two strains of N. australe were examined in this study. The strain BL24 of N. australe (haplotype H1) was originally isolated from a symptomatic branch of Phoenicean juniper collected in a natural area on Caprera Island (Italy). The strain BOT48 of N. australe (haplotype H4) was isolated from wood tissues of symptomatic grapevine cordon collected in a vineyard in northern Sardinia.9 Both strains were identified on the basis of morphological characters and analysis of internal transcribed spacer (ITS) rDNA. Fungal DNA extraction, PCR amplification reactions, and DNA sequencing were carried out as reported by Linaldeddu et al.9 Representative sequences of both isolates were deposited in GenBank: BL24 (ITS: accession number JX312356); BOT48 (ITS: accession number HQ011406). Pure cultures of both strains were maintained on potato-dextrose agar (Fluka, Sigma-Aldrich Chemic GmbH, Buchs, Switzerland) and stored at 4 °C in the collection of the Dipartimento di Agraria, Sezione di Patologia Vegetale ed Entomologia, University of Sassari, Italy. Extraction and Purification of Phytotoxins from N. australe Strain BOT48. The fungus was grown in 2 L Erlenmeyer flasks containing 400 mL of Czapek medium amended with corn meal (pH 5.7). Each flask was seeded with 5 mL of a mycelia suspension and then incubated at 25 °C for 4 weeks in darkness. The culture filtrates (5.7 L) were acidified to pH 4 with 2 M HCl and extracted exhaustively with EtOAc. The organic extracts were combined, dried with Na2SO4, and evaporated under reduced pressure to give a brownred oil residue (4 g), having a high phytotoxic activity on grapevine leaves by the puncture leaf bioassay at 4 mg/mL. The residue was submitted to a bioassay-guided fractionation through column 1789



Article

[6, 1.5 mg, 0.2 mg/L Rf 0.24, eluent CHCl3/i-PrOH (95:5), Rf 0.65, eluent EtOH/H2O (6:4)]. Botryosphaerone D (5): [α]25D +18 (c 0.2, MeOH); ECD (c 5.16 × 10−4 M) Δε +17.7 (280), +8.5 (239), −46.2 (214); UV λmax nm (log ε) 222 (4.27), 288 (4.19); IR νmax cm−1 3307, 1622, 1310, 1126 [for the botryosphaerone D,7 [α]25D +13 (c 2.0, MeOH); UV (MeOH) λmax nm (log ε) 230 (2.19), 297 (2.78); IR (KBr) νmax cm−1 3428, 1626, 1311, 1126]; 1H and 13C NMR (CD3OD) data were very similar to those already reported;7 ESIMS (−) m/z 251 [M − H]−, 233 [M − H2O − H]−, APCIMS (+) m/z 253 [M + H]+. 3,4,8-Trihydroxy-6-methoxy-3,4-dihydro-1(2H)-naphthalenone (6): [α]25D +53 (c 0.2, MeOH); ECD (c 5.8 × 10−4 M) +11.1 (280), +7.6 (238), −38.6 (214); UV λmax nm (log ε) 214 (4.26), 230 (4.09), 238 (4.04), 280 (4.14), 317 (3.79); IR νmax cm−1 3384, 1739, 1625, 1362, 1169 (Pittayakhajonwut et al.:30 [α]25D +49.38 (c 0.048, MeOH); UV (MeOH) λmax nm (log ε) 216 (4.21), 230 (4.04); 237 (3.95), 281 (4.10), 321 (3.82); IR (KBr) νmax cm−1 3494, 3405, 2958, 2927, 2855, 1729, 1636, 1578, 1459, 1432, 1403, 1375, 1360, 1291, 1164, 1128, 1075, 1031, 843); 1H and 13C NMR data were very similar to those already reported;30 ESIMS (−) m/z 223 [M − H]−, 207 [M − OH]−; APCIMS (+) m/z 225 [M + H]+. Phytotoxic Activity. The organic extracts and the pure compounds were assayed on holm oak (Quercus ilex L.), cork oak (Q. suber L.), and grapevine leaves (Vitis vinifera L.) by leaf puncture assay. The organic extracts were tested at 1, 2, and 4 mg/mL. Pure compounds (1−6) were assayed at 1, 0.5, 0.25, and 0.125 mg/mL. Extracts, fractions, and compounds were dissolved in MeOH and diluted with distilled H2O up to the assay concentrations (the final content of MeOH was 4%). Droplets of the test solutions (20 μL) were applied on the axial side of leaves that had previously been needle punctured. Droplets (20 μL) of MeOH in distilled H2O (4%) were applied on leaves as control. Each treatment was repeated twice. The leaves were then kept in a moist chamber to prevent the droplets from drying. Leaves were observed daily and scored for symptoms after 1 week. The effect of the toxins on the leaves, consisting of necrotic spots surrounding the puncture, was observed up to 15 days. Lesions were estimated using APS Assess 2.0 software42 following the tutorials in the user’s manual. The lesion size was expressed in mm2. Cyclobotryoxide (1) and tyrosol (4) were also tested on detached leaves of grapevine cv. Cannonau (red vine). The leaves with their petioles were immersed in 1 mL of toxic solution until its complete absorption and then transferred to distilled H2O. Toxicity symptoms were recorded 48 h later. Both compounds were tested at 0.25 and 0.5 mg/mL.

■

(4) Amponsah, N. T.; Jones, E. E.; Ridgway, H. J.; Jaspers, M. V. Aust. Plant Dis. Notes 2009, 4, 6−8. (5) Espinoza, J. G.; Briceño, E. X.; Chávez, E. R.; Ú rbez-Torres, J. R.; Latorre, B. A. Plant Dis. 2009, 93, 1187−1194. (6) Inderbitzin, P.; Bostock, R. M.; Trouillas, F. P.; Michailides, T. J. Mycologia 2010, 102, 1350−1368. (7) Xu, Y. X.; Lu, C. H.; Zheng, Z. H.; Shen, Y. N. Helv. Chim. Acta 2011, 94, 897−902. (8) Lazzizera, C.; Frisullo, S.; Alves, A.; Phillips, A. J. L. Plant Pathol. 2008, 57, 948−956. (9) Linaldeddu, B. T.; Scanu, B.; Schiaffino, A.; Serra, S. Phytopathol. Mediterr. 2010, 49, 417−420. (10) Sakalidis, M. L.; Hardy, G. E. J., St.; Burgess, T. I. Australas. Plant Pathol. 2011, 40, 510−521. (11) Evidente, A.; Sparapano, L.; Fierro, O.; Bruno, G.; Giordano, F.; Motta, A. Phytochemistry 1998, 48, 1139−1143. (12) Evidente, A.; Maddau, L.; Spanu, E.; Franceschini, A.; Lazzaroni, S.; Motta, A. J. Nat. Prod. 2003, 66, 313−315. (13) Sparapano, L.; Bruno, G.; Fierro, O.; Evidente, A. Phytochemistry 2004, 65, 189−198. (14) Evidente, A.; Masi, M.; Linaldeddu, B. T.; Franceschini, A.; Scanu, B.; Cimmino, A.; Andolfi, A.; Motta, A.; Maddau, L. Phytochemistry 2012, 77, 245−250. (15) Venkatasubbaiah, P.; Sutton, T. B.; Chilton, W. S. Phytopathology 1991, 81, 243−247. (16) Martos, S.; Andolfi, A.; Luque, J.; Mugnai, L.; Surico, G.; Evidente, A. Eur. J. Plant Pathol. 2008, 121, 451−461. (17) Evidente, A.; Punzo, B.; Andolfi, A.; Cimmino, A.; Melck, D.; Luque, J. Phytopathol. Mediterr. 2010, 49, 74−79. (18) Djoukeng, J. D.; Polli, S.; Larignon, P.; Abou-Mansour, E. Eur. J. Plant Pathol. 2009, 124, 303−308. (19) Pretsch, E.; Bühlmann, P.; Affolter, C. Structure Determination of Organic Compounds-Table of Spectral Data, 3rd ed.; Spinger-Verlag: Berlin, 2000; pp 161−243, 385−404. (20) Nakanishi, K.; Solomon, P. H. Infrared Absorption Spectroscopy, 2nd ed.; Holden Day: Oakland, 1977; pp 17−44. (21) Berger, S.; Braun, S. 200 and More Basic NMR Experiments: A Practical Course, 1st ed.; Wiley-VCH: Weinheim, 2004. (22) Breitmaier, E.; Voelter, W. Carbon-13 NMR Spectroscopy; VCH: Weinheim, 1987; pp 183−280. (23) Mennucci, B.; Claps, M.; Evidente, A.; Rosini, C. J. Org. Chem. 2007, 72, 6680−6691. (24) Ahamad, P. Y. A.; Kunhi, A. A. M.; Divakar, S. J. Microbiol. Biotechnol. 2001, 17, 371−377. (25) Shen, Y. Arch. Environ. Contam. Toxicol. 1998, 34, 229−34. (26) Barreto, G. E. S.; de Oliveira, E. S.; de Villar, F. A. G. A.; dos Santos, G. S.; Lima, R. M. F.; Costa, S. L.; de Fatima Dias Costa, M.; Egito, E. S. T.; El-Bacha, R. S. Braz. Archiv. Biol. Technol. 2007, 50, 839−849. (27) Nakai, N.; Murata, M.; Nagahama, M.; Hirase, T.; Tanaka, M.; Fujikawa, T.; Nakao, N.; Nakashima, K.; Kawanishi, S. Free Radical Res. 2003, 37, 69−76. (28) Li, X.; Yao, Y.-H.; Zheng, Y.-N.; Lin, W.-H.; Sattler, I.; Deng, Z.W. Zhongguo Tianran Yaowu 2007, 5, 20−23. (29) Capasso, R.; Cristinzio, G.; Evidente, A.; Scognamiglio, F. Phytochemistry 1992, 31, 4125−4128. (30) Pittayakhajonwut, P.; Sohsomboon, P.; Dramae, A.; Suvannakad, R.; Lapanun, S.; Tantichareon, M. Planta Med. 2008, 74, 281−286. (31) Evidente, A.; Maddau, L.; Scanu, B.; Andolfi, A.; Masi, M.; Motta, A.; Tuzi, A. J. Nat. Prod. 2011, 74, 757−763. (32) Andolfi, A.; Mugnai, L.; Luque, J.; Surico, G.; Cimmino, A.; Evidente, A. Toxins 2011, 3, 1569−1605. (33) Cimmino, A.; Villegas-Fernández, A. M.; Andolfi, A.; Melck, D.; Rubiales, D.; Evidente, A. J. Agric. Food Chem. 2011, 59, 9201−9206. (34) Van Niekerk, J. M.; Crous, P. W.; Fourie, P. H.; Halleen, F. Mycologia 2004, 4, 781−798. (35) Taylor, A.; Hardy, G. E. J., St.; Wood, P.; Burgess, T. Australas. Plant Pathol. 2005, 34, 187−195.

AUTHOR INFORMATION

Corresponding Author

*Tel: +39 081 2539178. Fax: +39 081 2539186. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The NMR spectra were recorded in the laboratory of Chimica Biomolecolare del CNR, Pozzuoli. This work was supported in part by grants from Fondazione Banco di Sardegna, Parco Nazionale dell'Arcipelago di La Maddalena and Italian Ministry of University and Research. Contribution DISSPAPA N. 273.

■

REFERENCES

(1) Slipper, B.; Fourie, G.; Crous, P. W.; Coutinho, T. A.; Wingfield, B. D.; Wingfield, M. J. Mycologia 2004, 96, 1030−1104. (2) Burgess, T. I.; Sakalidis, M. L.; Hardy, G. S. J. Aust. Ecol. 2006, 31, 559−566. (3) Armengol, J.; Gramaje, D.; Pérez-Sierra, A.; Landeras, E.; Alzugaray, R.; Martos, S.; Luque, J. Plant Dis. 2008, 92, 980. 1790



Article

(36) Apoga, D.; Åkesson, H.; Jansson, H. B.; Odham, G. Eur. J. Plant Pathol. 2002, 108, 519−526. (37) Reino, J. L.; Hernández-Galán, R.; Durán-Patron, R.; Collado, I. G. J. Phytopathol. 2004, 152, 563−566. (38) Strange, R. N. Nat. Prod. Rep. 2007, 24, 127−144. (39) Mahoney, N.; Molyneux, R. J.; Smith, L. R.; Schoch, T. K.; Rolshausen, P. E.; Gubler, W. D. J. Agric. Food Chem. 2005, 53, 8148− 8155. (40) Albuquerque, P.; Casadevall, A. Med. Mycol. 2012, 50, 337−345. (41) Chen, H.; Fujita, M.; Feng, Q.; Clardy, J.; Fink, G. R. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 5048−5052. (42) Lamari, L. Assess Image Analysis Software for Plant Disease Quantification; APS Press: St. Paul, MN, 2002.

1791


Cyclobotryoxide, a Phytotoxic Metabolite Produced by the Plurivorous

Recommend Documents