Mitidjospirone, a new spirodioxynaphthalene and GC-MS screening of secondary metabolites produced by strains of Lasiodiplodia mitidjana associated to Citrus sinensis dieback

Abstract Mitidjospirone, a new spiridioxynaphthalene, was isolated from the mycelial extract of a strain of Lasiodiplodia mitidjana, a recently described species belonging to the family Botryosphaeriaceae. Its structure was elucidated by extensive spectroscopic analysis and the absolute configuration was determined by electronic circular dichroism (ECD) experiment. Furthermore, several known compounds were identified during the screening of secondary metabolites produced by four strains of L. mitidjana. Graphical Abstract


Introduction
Botryosphaeriaceae is a genus rich family containing numerous species with a cosmopolitan distribution and a large variety of plant hosts, such as grapevine, eucalyptus, citrus, mango and coconut (Slippers and Wingfield 2007;Mehl et al. 2017). Lasiodiplodia is one of the most common genera, which is particularly diffused in tropical and subtropical regions, where it is responsible of a variety of plant diseases (Phillips et al. 2013). For this reason, in the last decades, the literature regarding the associations between species of Lasiodiplodia and plants has been substantially enriched. Considerable attention has been given to Lasiodiplodia theobromae, which is the most representative species of this genus ( Urbez-Torres 2011) and this species turned out to be a good source of biotechnologically relevant products such as enzymes (F elix, Lib orio, et al. 2018), polysaccharides (Selbmann et al. 2003), and secondary metabolites . In this respect, the capacity to produce secondary metabolites deserves particular attention because these compounds are directly involved in host-fungus interactions Salvatore, Andolfi, and Nicoletti 2020).
During the study of the impact of Botryosphaeriaceae species on citrus trees in Algeria, strains of the novel species Lasiodiplodia mitidjana were isolated from necrotic tissues of citrus branch canker. In the present study, a screening of secondary metabolites of four strains of L. mitidjana isolated from Citrus sinensis was carried out for the first time. This study led to the isolation of a new spirodioxynaphthalene, namely mitidjospirone (1), along with several known compounds, such as lasiodiplodin, indole-3-carboxylic acid (ICA), indole-3-acetic acid (IAA), tyrosol, eugenol and succinic acid.

Results and discussion
Four isolates of L. mitidjana (Berraf-Tebbal et al. 2020), from citrus trees showing symptoms of dieback in Algeria ( Figure S1 in Supplementary materials), were screened for their ability to produce secondary metabolites in vitro. All isolates were grown at 25 C for 21 days on Czapek medium amended with 2% cornmeal. After incubation, the culture broth and the mycelium of each isolate were separated by filtration and extracted with organic solvents as reported in the Experimental section. Culture filtrate and mycelial crude extracts were analysed via GC-MS after derivatization with N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA). A number of metabolites is produced in sufficient amounts to be directly detected and identified in the crude extracts (Table S1 in Supplementary materials). Many compounds typical of Lasiodiplodia species were identified (e.g., (3R,4R)-4-hydroxymelleins, IAA, ICA, lasiodiplodin, (R)-mellein, tyrosol)  and the comparative study of crude extracts allowed to show the variability of metabolites among strains. Some metabolites were identified comparing their EI mass spectra with known substances present in NIST 14 mass spectral library. Moreover, the identification process was implemented by using our custom MS target library which collect the full set of mass spectra of trimethylsilyl metabolites acquired during our previous research works on strains of L. theobromae (F elix, F elix et al. 2019;. Different metabolic profiles of L. mitidjana strains suggest that the production of secondary metabolites is strain dependent confirming data previously reported for other species of Lasiodiplodia (Table S1) (Andolfi et al. 2014(Andolfi et al. , 2016F elix, Salvatore, et al. 2018).
The most productive strain is ALG 111 ( Figure S2 in Supplementary materials). This strain produces an ample variety of metabolites known as phytohormones or for their bioactivities. Among them, ICA was reported as phytotoxic and cytotoxic compound produced by strains of L. theobromae grown in different cultural conditions (F elix, F elix et al. 2019; and it was considered responsible of fungal infections. Furthermore, ICA was detected in the culture filtrate extract along with IAA suggesting that it was biosynthesised from tryptophan via the IAA pathway (Salvatore, Alves, and Andolfi 2020).
(R)-Mellein is present in all the crude extracts under examination except for the crude extracts of ALG 39. (R)-Mellein belongs to the family of 3,4-dihydrocoumarins (Saeed 2016) and is commonly produced by botryosphaeriaceous fungi Salvatore et al. 2021).
Mycelial extracts showed the presence of some fatty acids, such as palmitic, linoleic and linolenic acids, which are constituents of the fungal cell membranes (Vestal and White 1989) (Table S1).
Some compounds were not detectable directly in the crude extracts, but they were identified after fractionation by column chromatography of the crude extracts as reported in Table S2 (Supplementary materials). In particular, the GC-MS analysis of the chromatographic fractions of the culture filtrate extract of ALG 111 showed the presence of some additional compounds: 1,8 dihydroxyantraquinone, tyrosol, phenylethanol, protocatechuic, vanillic, syringic and cinnamic acids. (R)-Mellein, phenylethanol and (3R,4R)-4-hydroxymellein were identified in the chromatographic fractions obtained from ALG 39 extract.
Two spirodioxynaphthalenes (1 and 2, Figure 1) were isolated by a chromatographic purification process of the mycelial extract of ALG 111 and their structures elucidated using spectroscopic methods.
1D and 2D NMR spectra of 1 were recorded in deuterated methanol (CD 3 OD) on Bruker spectrometers and the same solvent was used as internal standard. NMR data In fact, the optical rotation of compound 1 recorded in acetone is [a] 25 D þ245 , while the optical rotation of palmarumycin BG3 is À261 . Furthermore, CD bands of compound 1 at 221 and 206 nm (De 21.47 and À23.24) turn out to be the mirror images of the ones at 227 and 197 nm (De À22.34 and 17.20) of palmarumycin BG3 (Cai et al. 2011). Hence, the absolute configuration (1S,3S) was assigned to compound 1. This is the first report concerning the isolation and identification of this compound and it was named mitidjospirone (1). 1 H spectrum of 2 is very similar to the one acquired for 1 ( Figure S10 in Supplementary materials). In fact, it showed the typical signal pattern at d 7.60-6.96 assigned to the 1,8-dioxynaphthalene moiety and to the 1,2,3-trisubstituted aromatic ring (Kouam et al. 1993). Furthermore, the X-ray crystal structure analysis performed on a single crystal allowed to unambiguously identify compound 2 as palmarumycin JC1, which structure and relative configuration is already known ( Figure S11 and Table  S3 in Supplementary materials) (Prajoubklang et al. 2005). Both compounds (1 and 2) belong to two-oxygen-bridge-type spirodioxynaphthalenes, which is a group of fungal secondary metabolites consisting of a 1,8-dihydroxynaphthalene-derived spiroketal unit linked to a second oxidized naphthalene moiety, that shows a great variety of biological activities (Cai et al. 2010).
Concerning the possible biosynthetic origin, it was hypothesized that these polyketidic compounds derive from 1,8-dihydroxynaphthalene (DHN), and that palmarumycin CP1 is produced as intermediate through the oxidative coupling (Cai et al. 2010).
To our knowledge, among Lasiodiplodia species, only an endophytic strain of L. theobromae isolated from fresh healthy leaves of Vitex pinnata was reported as producer of spirodioxynaphthalenes. In particular, cladospirone B was isolated and identified, along with other putatively identified compounds of this group (Kamal et al. 2017).

General experimental procedures
Optical rotations of pure metabolites were measured on a Jasco polarimeter (Tokyo, Japan). The UV-Vis spectra were recorded from 200 to 600 nm (optical path 1.0 cm) at 25.0 C, under a constant flow of nitrogen with Cary model 5000 Spectrophotometer by Varian C. (Palo Alto, CA, USA) as well as the far UV-ECD spectra were recorded with a Jasco CD spectrometer model J-715, from 200 to 600 nm (optical path 1.0 cm) at 25.0 C, under a constant flow of nitrogen. For both measurements, the blank was represented by MeOH.
1 H and 13 C NMR spectra were recorded at 400 and 100 MHz, respectively, in deuterated methanol (CD 3 OD), on Bruker (Karlsruhe, Germany) spectrometers and the same solvent was used as internal standard. Analytical TLC were performed on silica gel plates (Kieselgel 60, F254, 0.25, Merck, Darmstadt, Germany). The spots were visualised by exposure to UV radiation (253 nm), or by spraying first with 10% H 2 SO 4 in methanol followed by heating at 110 C for 10 min. Chromatography was performed on silica gel column (Merck, Kieselgel 60, 0.063-0.200 mm).

Fungal isolation and identification
Lasiodiplodia mitidjana strains were isolated from citrus trees showing canker and dieback symptoms. These strains have been described as a novel taxon in our previous work (

Fermentation conditions
Mycelial plugs from stock cultures maintained on potato dextrose agar (Oxoid) were used to inoculate 500 mL of Czapek Dox broth (Oxoid) added with 2% cornmeal in 1-L Erlenmayer flasks. The cultures were incubated on stationary phase in the dark at 25 C. After 21 days, the liquid phase was separated by filtration on Whatman No. 5 filter paper, and the culture filtrates stored at À20 C.

Extraction and isolation of metabolites from mycelia
Fresh mycelia of L. mitidjana strains were extracted as previously described with slight modifications . Briefly, each mycelium was homogenised in a mixer with 440 mL of MeOH-H 2 O (NaCl 1%) mixture (55:45, v/v). The suspension was centrifuged (40 min at 7000 rpm, 10 C) and separated from the supernatant. A second homogenization with 250 mL of the mixture reported above was performed followed by centrifugation. The supernatant of the second extraction was combined with the first supernatant for the subsequent extraction (3 times) with CHCl 3 . The organic extracts were combined, dried on anhydrous Na 2 SO 4 , and evaporated under reduced pressure yielding mycelial crude extract (ME) as yellowish oil (ME ALG111 ¼113.7 mg, ME ALG39 ¼96.8 mg, ME ALG34 ¼30.8 mg, ME ALG81 ¼24.5 mg).
Mycelial extracts were purified by column chromatography (CC) on silica gel, yielding the identified metabolites. Each chromatographic fraction was analysed via GC-MS after derivatization with BSTFA. Several metabolites were already detected during the screening of metabolites present in crude extracts, but the purification allowed to concentrate and identify some additional metabolites. In order to determine the stereochemistry, (R)-mellein was submitted to optical rotation experiments . ME ALG111 was fractionated by CC on silica gel (40 cm Â 1.5 cm i.d.) eluted with CHCl 3 /i-PrOH (9:1, v/v), originating 9 homogeneous fractions. The last fraction was eluted with methanol (A 6.2 mg, B 7.1 mg, C 3.9 mg, D 11.1 mg, E 24.9 mg, F 25.4 mg, G 3.9 mg, H 5.6 mg, I 22.5 mg). Fraction C was identified as (3R,4R)-4-hydroxymellein. Fraction F was purified by TLC on silica gel eluted with EtOAc/n-hexane (6:4, v/v) giving 1 (1.8 mg as amorphous solid, R f 0.57, in the same chromatographic conditions) and 2 (2.5 mg as crystalline solid, R f 0.43 in the same chromatographic conditions). ME ALG39 was fractionated by CC on silica gel (40 cm Â 1.5 cm i.d.) eluted with CHCl 3 /i-PrOH (9:1, v/v) originating 8 homogeneous fractions. The last fraction was eluted with methanol (A 4.2 mg, B 6.2 mg, C 4.0 mg, D 9.1 mg, E 25.8 mg, F 6.3 mg, G 4.2 mg, H 27.8 mg). ME ALG34 was fractionated by CC on silica gel (30 cm Â 1.5 cm i. d.) eluted with CHCl 3 /i-PrOH (9:1, v/v) originating 5 homogeneous fractions. The last fraction was eluted with methanol (A 1.2 mg, B 4.7 mg, C 2.5 mg, D 6.4 mg, E 14.5 mg). ME ALG81 was fractionated by CC on silica gel (30 cm Â 1.5 cm i.d.) eluted with CHCl 3 /i-PrOH (9:1, v/v) originating 5 homogeneous fractions. The last fraction was eluted with methanol (A 0.8 mg, B 2.7 mg, C 2.9 mg, D 5.6 mg, E 11.7 mg).

GC-MS analyses
GC-MS data were acquired on crude extracts and chromatographic fractions after trimethylsilylation with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (Fluka, Buchs, Switzerland). Samples were derivatized and analysed according to a previously reported procedure (Guida et al. 2015).
GC-MS measurements were performed with an Agilent 6850 GC (Milan, Italy), equipped with an HP-5MS capillary column (5% phenyl methyl poly siloxane stationary phase), coupled to an Agilent 5973 Inert MS detector operated in the full scan mode (m/z 29-550) at a frequency of 3.9 Hz and with the EI ion source and quadrupole mass filter temperatures kept, respectively, at 200 C and 250 C. Helium was used as carrier gas at a flow rate of 1 mLÁmin À1 . The injector temperature was 250 C and the temperature ramp raised the column temperature from 70 C to 280 C: 70 C for 1 min; 10 CÁmin À1 until reaching 170 C; and 30 CÁmin À1 until reaching 280 C. Then, it was held at 280 C for 5 min. The solvent delay was 4 min.
Metabolites were identified by comparing their EI mass spectra at 70 eV with spectra of known substances present in our custom MS target library (F elix, F elix et al. 2019;) and in the NIST 14 mass spectral library (NIST 14). Moreover, the identification was supported by Kovats retention index (RI) calculated for each analyte by the Kovats equation, using the standard n-alkane mixture in the range C7-C40 (Sigma-Aldrich, Saint Louis, MO, USA) .
The relative percentage of each compound is defined by the area of selected peak divided for the sum of the peak area.
3.7. X-ray crystal structure analysis of compound 2 Single crystals of 2 suitable for X-ray structure analysis were obtained by slow evaporation of MeOH solution. One selected crystal was mounted at ambient temperature on a Bruker-Nonius KappaCCD diffractometer (graphite monochromated MoKa radiation, k ¼ 0.710 73 Å, CCD rotation images, thick slices, and u and x scans to fill the asymmetric unit). The structure was solved by direct methods using SIR97 program (Altomare et al. 1999). All non-H atoms were anisotropically refined by the full matrix least squares method on F 2 against all independent measured reflections using the SHELXL-2018/3 program (Sheldrick 2015). A part hydroxy hydrogens that were located in difference Fourier maps, all H atoms were geometrically positioned and isotropically refined according to a riding model. Due to the absence of strong scatterer atoms, it was not possible to assign the absolute configuration. Figure S11 in Supplementary Materials was generated by ORTEP-3 program (Farrugia 2012). Crystal data and structure refinement parameters are reported in the Table S3 in Supplementary Materials.

Conclusions
In summary, the chemical investigation of culture filtrate and mycelial extracts of four strains of L. mitidjana isolated from citrus trees affected by dieback showed that the occurrence of secondary metabolites is strain dependent. Several known metabolites, typically detected as products of Lasiodiplodia species, were identified (e.g., ICA, lasiodiplodin, (R)-mellein). Furthermore, two unusual compounds belonging to spirodioxynaphthalenes were isolated from the mycelial extract of the most productive strain (ALG 111). This is the first report dealing with the identification of mitidjospirone.