Bioactive specialized metabolites produced by the emerging pathogen Diplodia olivarum

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Introduction
Diplodia Fr. is a large genus in the family Botryosphaeriaceae typified by Diplodia mutila (Fr.: Fr.) Fr. [1]. Species of Diplodia are cosmopolitan in temperate and subtropical regions and occur on a wide range of angiosperm and gymnosperm hosts [2]. They exhibit diverse life-styles spanning from endophytes able to inhabit different asymptomatic plant tissues to aggressive pathogens that cause severe diseases in various plant hosts [2][3][4][5]. The increasing number of reports of new diseases caused by these pathogens during the last decades has stimulated the research into the virulence factors involved in the pathogenesis process. This allowed several bioactive secondary metabolites to be isolated and identified belonging to different classes of organic compounds such as pimarane diterpenoids, α-pyrones, furanones, diplobifuranylones, naphthoquinones, biphenols, cyclohexene oxides, furopyrans, isochromanones and melleins from the emerging pathogens
Recently, another species namely Diplodia olivarum has been emerging as an aggressive pathogen on different plant hosts in Italy. D. olivarum was originally found on rotting olive drupes in southern Italy and described as a new species in 2008 [7]. It was later reported as a canker agent on carob tree [8], lentisk [9] and wild olive [10]. Symptoms of the disease in infected hosts include sunken cankers with characteristic wedge-shaped wood necrosis on branches and stem. Foliar symptoms have also been observed especially on lentisk shoots ( Figure 1). Therefore, given the growing expansion of severe dieback caused by D. olivarum in several natural ecosystem in Italy, and the still limited information available about the bioactive specialized metabolites produced by this emerging pathogen, a study was conducted to isolate, identify and evaluate the phytotoxic, antifungal, antioomycetes and zootoxic activities of the main compounds produced in vitro.
Olicleistanone (1) has a molecular formula of C22H28O4 as deduced from its HR ESIMS spectrum and consistent with nine hydrogen deficiencies. Preliminary investigation of its 1 H and 13 C NMR spectra (Table 1) showed that it is close related to a tricyclic nor-diterpenoid, with aromatized and cyclohexadiene rings (A and B) joined to a dihydropyran ring (D) generated probably from a cleistanthane carbon skeleton [19]. The signal at 195.5 in the 13 C NMR spectrum also suggested the presence of a conjugated ketone group [20]. These results are in full agreement with the bands typical for carbonyl and aromatic groups observed in the IR spectrum [21] and the absorption maxima observed in the UV spectrum [22].
In particular, its 1 H and COSY spectra [23] showed the presence of two doublets (J = 7.9 Hz) at δ 7.31 and 7.18, typical signals of two ortho-coupled protons (H-11 and H-12) of a 1,2,3,4tetrasubstituted C benzene ring. The same spectra showed the singlets of a methoxy group (CH3- The long range couplings observed in the HMBC spectrum [23] (Table 1)  respectively. The resulting signal at  132.9 was assigned to C-9 [20]. Furthermore, the correlation between C-7 and H-20A allowed the ethoxy group to be located at C-7 and consequently the methoxy group at C-15.
Attempts to assign the relative configuration of 1 were made recording a NOESY spectrum.
The measured NOESY correlations are reported in Table 2 but since there is no clear correlation between the protons of the methoxy and ethoxy groups, these data alone were not enough to assign the relative configuration of the two chiral centres (C-7 and C-15). In order to better interpret NMR data, a molecular modelling study was undertaken. for the prediction of 13 C chemical shifts of flexible compounds [24]. For the two diastereomers, 6 or 10 conformers were found with detectable populations at room temperature. The various conformers differed in the conformation of the methoxy and ethoxy groups, but also in the puckering of ring A. A clear difference between the two diastereomers was the orientation of H-15, which was predominantly pseudo-equatorial in (7S,15S)-1 and pseudo-axial (7S,15R)-1. Thus, we thought that the coupling constants between H-15 and H-16a/H-16b could be used to discriminate between the two isomers. Experimentally, H-15 appears as a doublet with splitting of 3.3 Hz, meaning that one J15/16 was small (3.3 Hz) and the other one negligible. This fact was in accordance with a pseudo-equatorial orientation. 3 J15/16 were then estimated with two different methods, namely a Karplus curve and spin-spin coupling calculations at B3LYP/pcJ-0 level. The results are shown in Table 3 and strongly support the assignment of 1 as (7S*,15S*)-1. Finally, 13 C-NMR calculations were run at the B3LYP/6-31G(d) level. The estimated rms (root-mean-square) error between experimental and calculated 13 C chemical shifts was acceptable (2.4-2.5) but similar for both isomers, thus confirming the above assignment but without further supporting it. Anyway, we believe that our argument based on J-couplings is accurate enough to assign the relative configuration.
Coming to the absolute configuration, we measured the ECD spectrum of a solution of 1 in acetonitrile (1 mM, 0.01 cm cell). To our surprise, the ECD spectrum was not distinguishable from the baseline over the whole range (185-400 nm, data not shown), despite the optimal absorption (0.3 to 0.8 for the absorption peaks). We must conclude that the isolated sample of 1 was a racemate. Racemic natural products are rare but sometimes encountered and are thought to be the result of nonenzymatic reactions [25]. We noticed that the chirality center at C-7 of 1 is a tertiary benzylic carbon in α position to carbonyl group; however, racemization of this centre does not occur in a post-synthetic step, otherwise one would obtain a couple of diastereomers. On the other hand, the isolated (7S,15S)-1 isomer is more stable than its (7S,15R) diastereomer by about 2 kcal/mol at our level of calculation, suggesting that if the chiral center at C-15 is biosynthesized in a later step than C-7, its configuration would be dictated by that at C-7.
Cleistanthane-type diterpenoids are produced by different fungal and plant species but few examples are available on cleistanthane nor-diterpenoids as aspergiloids A, B, F and G isolated from the fermentation broth extract of Aspergillus sp. YXf3, an endophytic fungus from Ginkgo biloba. However, no biological activities were reported for these compounds [26,27].
Sphaeropsidins A, C and G, as well as the other ones B, D-F previously isolated from D.
Smardesines and chenopodolins belong also to the same group. These compounds were isolated as cytotoxic and phytotoxic metabolites from Smardaea sp. AZ0432 living in the moss Ceratodon purpureus [30] and Phoma chenopodiicola, a fungus proposed for the biocontrol of Chenopodoium album [31,32]. Sphaeropsidin A and its 6-O-acetyl derivative also showed interesting antimicrobial [33] and anticancer activity [2,34,35].
Melleins are 3,4-dihydroisocoumarins mainly produced by many fungi of various genera but also by plants, insects and bacteria. They possess several biological activities as phytotoxic, zootoxic and antifungal effects [36]. (-)-Mellein was toxic on grapevine leaves and grapevine calli [2,37,38] and was detected in symptomatic and asymptomatic wood samples and in green shoots [37] on grapevines showing Botryosphaeria dieback and leaf stripe disease. Its role in pathogenesis was investigated by examining the extent to which it caused the expression of defense-related genes in grapevine calli [38]. Recently, (-)-mellein was also identified as a metabolites of Lasiodiplodia euphorbiaceicola during a screening of phytotoxic metabolites isolated from some Lasiodiplodia spp. infecting grapevine in Brazil [17] and from Sardiniella urbana, a pathogen found on declining European hackberry trees in Italy [39].

Conclusion
This study represents the first investigation on the secondary metabolites produced by D.

Experimental General experimental procedures
Optical rotations were measured in MeOH on a P-1010 digital polarimeter (Jasco, Tokyo, Japan), unless otherwise note. IR spectra were recorded as deposit glass film on a 5700 FT-IR spectrometer (Jasco, Tokyo, Japan) and UV spectra were measured in MeCN on a V-530 spectrophotometer (Easton, MD, U.S.A.); 1 H and 13 C NMR spectra were recorded at 400 and 100 MHz in CDCl3 on Bruker spectrometer (Billerica, MA, U.S.A.). The same solvent was used as internal standard. The multiplicities were determined by DEPT spectrum [23]. COSY, HSQC, HMBC and NOESY spectra were recorded using Bruker microprograms. HR ESIMS spectra were recorded on a 6120 Quadrupole LC/MS instrument (Agilent Technologies, Milan, Italy). Analytical and preparative TLC were performed on silica gel (Kieselgel 60, F254, 0.25 and 0.5 mm respectively) and on reversed phase (Kieselgel 60 RP-18, F254, 0.20 mm) plates (Merck, Darmstadt, Germany). The spots were visualized by exposure to UV radiation (253 nm), 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).

Production, extraction and purification of secondary metabolites
The fungus was grown on two different liquid media (Czapek amended with 2% yeast extract and mineral salt medium [41] both at pH 5.7) in 1L Erlenmeyer flasks containing 250 mL medium. Each flask was seeded with 5 mL of a mycelial suspension and then incubated for 30 d at 25 °C. Culture filtrates were obtained by filtering the culture through filter paper in a vacuum system. The culture filtrate (14.5 L), obtained growing the fungus on Czapek medium, was acidified to pH 4 with 2 N HCl and extracted exhaustively with EtOAc. The combined organic extracts were dried with Na2SO4 and evaporated under reduced pressure. The brown-red oil residue recovered (4.5 g) was fractioned by column chromatography on silica gel (90 × 4 cm) eluted with n-hexane-EtOAc (7:3). Eleven fractions were collected and pooled on the basis of similar TLC profiles. Olicleistanone (1)

Leaf puncture assay
Phaseolus vulgaris L, Juglans regia L. and Quercus suber L. leaves were used for this assay.
Each compound was tested at 1.0 mg/mL. The assay was performed as previously reported [14].
Each treatment was repeated three times. Leaves were observed daily and scored for symptoms 14 after 5 days. The effect of the toxins on the leaves was observed up to 10 days. Lesions were estimated using APS Assess 2.0 software following the tutorials in the user's manual. The lesion size was expressed in mm 2 .

Antifungal assays
All compounds (1-6) were preliminarily tested on four different plant pathogens including two fungal species (Athelia rolfsii and Diplodia corticola) and two oomycetes (Phytophthora cambivora and P. lacustris). The sensitivity of all species to these compounds was evaluated, depending on the species, on CA (carrot agar medium) or PDA (potato dextrose agar) as inhibition of the mycelial radial growth. The assay was performed as previously reported [42]. Each metabolite was tested at 200 μg/plug. Methanol was used as negative control. Metalaxyl-M (mefenoxam; p.a. 43.88%; Syngenta), a synthetic fungicide to which the oomycetes are sensitive, and PCNB (pentachloronitrobenzene) for ascomycetes and basidiomycetes, were used as positive control. Each treatment consisted of three replicates and the experiment was repeated twice.

Artemia salina bioassay
All compounds were assayed on brine shrimp larvae (Artemia salina L.). The assay was performed in cell culture plates with 24 cells (Corning) as previously described [14. The metabolites were tested at 100 g/mL. Tests were performed in quadruplicate. The percentage of larval mortality was determined after 36 h incubation at 27 °C in the dark.

Supporting Information
1D and 2D NMR data for 1 and HRESI-MS spectra of 1-3.