Xerophytic Lichens from Gypsiferous Outcrops of Arid Areas of Andalusia as a Source of Anti-Phytopathogenic Depsides

In a survey to evaluate the potential of lichens associated with gypsum areas as sources of new antifungal metabolites, six species of lichens were collected in the gypsum outcrops of the Sorbas Desert (Diploschistes ocellatus and Seirophora lacunosa) and the Tabernas Desert (Cladonia foliacea, Acarospora placodiformis, Squamarina lentigera and Xanthoparmelia pokornyi) in southern Spain. Raw lichen acetone extracts were tested against a panel of seven phytopathogenic fungi, including Botrytis cinerea, Colletotrichum acutatum, Fusarium oxysporum f.sp cubense TR4, Fusarium ploriferaum, Magnaporthe grisea, Verticillium dahliae and Zymoseptoria tritici. Active extracts of Cladonia foliacea, Xanthoparmelia pokornyi and Squamarina lentigera were analyzed by HPLC-MS/MS and Molecular Networking to identify possible metabolites responsible for the antifungal activity. A total of ten depside-like metabolites were identified by MS/MS dereplication and NMR experiments, of which one was a new derivative of fumaroprotocetraric acid. The compounds without previously described biological activity were purified and tested against the panel of fungal phytopathogens. Herein, the antifungal activity against fungal phytopathogens of 4′-O-methylpaludosic acid, divaricatic acid and stenosporic acid is reported for the first time. Stenosporic and divaricatic acids displayed a broad antifungal spectrum against seven relevant fungal phytopathogens in a micromolar range, including the extremely resistant fungus F. oxysporum f. sp. cubense Tropical Race 4 (TR4). 4′-O-methylpaludosic acid exhibited specific antifungal activity against the wheat pathogen Z. tritici, with an IC50 of 38.87 µg/mL (87.1 µM) in the absorbance-based assay and 24.88 µg/mL (55.52 µM) in the fluorescence-based assay.


Introduction
Within all plant pathogens, the fungi kingdom is a highly diverse group that represents a devastating threat to plant health in agriculture, resulting in a negative economic impact worldwide with every single outbreak [1].Genetic plasticity allows fungi to quickly invade new hosts, as well as to develop resistance to traditional fungicidal substances used against these phytopathogens [2].Even with modern agriculture, fungal epidemics still occur in plants and generate substantial losses in terms of food production yields.For instance, back in 2016, the first known outbreak caused by the wheat blast fungus Magnaporthe oryzae in Asia devastated more than 15,000 hectares of wheat crops in Bangladesh.The available treatments could not contain the infection of the fungus, which strikes wheat heads, making it difficult for fungicides to reach the pathogen [3].Another example is Fusarium oxysporum f. sp.cubense Tropical Race 4 (TR4), an extremely resistant fungus that can persist in soil or on decaying host plant debris for up to 30 years in the form of chlamydospores and cause an outbreak in a Cavendish banana plantation in North Queensland, Australia, in March 2015 [4,5].In Europe, a new variety of stem-rust fungus attacked Sicilian crops back in 2016, destroying ten thousand hectares of crops and alarming researchers since this new strain displayed unusually high infection capabilities against plants resistant to disease [6].
Addressing this issue, the European Parliament developed Directive 2009/128/EC, related to the sustainable use of pesticides, and Regulation (EC) No. 1107/2009 on the marketing of phytosanitary products, both promoting the replacement of traditional chemical agents for phytosanitary treatments and other alternatives when possible.This last regulation was recently modified in Regulation (EU) 2017/1432, which established the use of novel low-risk natural substances as starting products for the development of new treatments, even when their half-life in the soil exceeds 60 days.These recent regulations open a new niche in the biodiscovery of substances with high-added value in the plant health market.This work is focused on the use of endemic gypsiferous lichens from southern Spain as a possible source for these substances, based on preliminary bioactivity results.
Lichens are symbiotic organisms born from the association of fungi and microalgae, which has allowed them to develop a complex metabolism with exceptional adaptation capabilities, allowing them to survive in hostile and desolated environments from the poles to the deserts [7].Among the mechanisms deployed for the adaptation of these organisms to harsh conditions, their response to oxidative stress [8], desiccation tolerance [9] and potential to produce secondary metabolites with antimicrobial properties can be highlighted [10][11][12].Lichens inhabiting gypsiferous fields are particularly rare species due to their chemical nature [13] and extremely hostile conditions [14] in such environments, and they have mainly been studied from a botanical point of view during the last forty years [15][16][17][18][19].For this reason, gypsiferous lichens are a poorly exploited source for the potential biodiscovery of new secondary metabolites with several applications.
Among the metabolites widely known to be produced by lichens, depsides are a class of polyphenols structurally featuring two or more monocyclic aromatic moieties linked by an ester bond.This class of compounds has previously shown antioxidant activity [20,21], antiproliferative activity on cancer cells [22,23], antibiofilm activity [24], antimicrobial activities on both bacteria and fungi [25,26], and some other relevant bioactivities [27].Compounds belonging to this structural class were already detected in the preliminary chemical analysis of the gypsiferous lichen extracts.
The objective of this research was the screening of xerophytic lichen samples from the Andalusian gypsiferous outcrops (Tabernas and Sorbas, Almeria province), followed by the isolation and identification of the bioactive compounds responsible for the antiphytopathogenic activity.

General
Extracts were analyzed by HPLC-UV-ESI-TOF on an Agilent 1200 RR, coupled to a Bruker maXis time-of-flight spectrometer with electrospray ionization, as reported by Martin et al. [28].Mass spectra were collected as full scans from 50 m/z to 2000 m/z.Data were analyzed using the platform available at Fundación MEDINA [29] and compared with the data available in Medina's In-House internal database and the Dictionary of Natural Products database.LC-UV-LRMS analyses were performed on an Agilent 1260 Infinity II (Agilent Technologies, Santa Clara, CA, USA) equipped with a single quadrupole LC-MS system.Mono-and bidimensional Nuclear Magnetic Resonance (NMR) spectra were recorded at 297K on a Bruker Avance III spectrometer (500 and 125 MHz for 1 H and 13 C, respectively) equipped with a 1.7 mm TCI MicroCryoProbeTM (Bruker Biospin, Fällanden, Switzerland).Chemical shifts from 1 H and 13 C were reported in parts per J. Fungi 2023, 9, 887 3 of 13 million using the signals of the residual solvents as internal reference (δ H 2.50 and δ C 39.52 ppm for DMSO-d 6 ).

Lichen Material
Samples of Diploschistes ocellatus var almeriensis and Seirophora lacunosa were collected from gypsiferous outcrops in Sorbas, Almeria, Spain, in October of 2020.Samples of Acarospora placodiiformis, Cladonia foliacea, Squamarina lentigera and Xanthoparmelia pokornyi were collected in Venta de los yesos, Tabernas Desert, Almeria, Spain, in October of 2020 (Table 1, Figure 1).The lichens were taxonomically identified by one of us (M.C.-P.), and reference samples were preserved in the University of Granada herbarium (GDA), with voucher codes provided in Table 1.

Extraction
The lichen samples were washed with distilled water and dried before their extraction.For the extraction, two grams of each sample were weighed and divided into two equal portions.One of the portions was extracted with 10 mL of acetone, and the other with 10 mL of a mixture of dichloromethane and methanol (1:1).All samples were extracted overnight by maceration.The resulting crude extracts were filtered, and the organic phase was evaporated under a nitrogen stream.Then, 5 mg of each sample was resuspended in 50 µL of DMSO to prepare the stock solution for the antifungal assays (100 mg/mL).Inactive extracts were not studied/analyzed further.

LC-MS/MS and Feature-Based Molecular Networking Analysis
LC-MS/MS acquisition was performed as previously described in the general section.Ionization of the eluting solvent was obtained using the standard maXis ESI source, adjusted to a drying gas flow of 11 L/min at 200 • C and a nebulizer pressure of 40 psig.The capillary voltage was set to 4000 V. Mass spectra were collected from 150 m/z to 2000 m/z in positive mode.The mass spectrometer was operated in data-dependent positive ion mode, automatically switching between full scan MS and MS/MS acquisitions.Full scan MS spectra (m/z 50-2000) were acquired in the TOF, and the top six most intense ions in a particular scan were fragmented using collision-induced dissociation (CID).
MS/MS spectra were converted to the mzXML file format by using Bruker Data Analysis software.The mzXML file was further processed with MZmine 2.539 following the described processing steps: mass detection, chromatogram builder, chromatogram deconvolution, isotopic peaks grouper, feature alignment, gap filling, filtering and direct submit/export for GNPS [30].The final processed file was then used for the online analysis on the GNPS platform (https://gnps.ucsd.edu/accessed on 25 July 2023), and Cytoscape 3.6.1 was used to visualize the generated molecular networks.FBMN data are available from the following link: https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=6cafd8eec333 427a81ccb5497bee4b7a accessed on 25 July 2023.

Crude Extract Fractionation
A total of 80 mg of each lichen extract was fractionated through a Gilson semiautomated HPLC-DAD system by creating a gradient on a column Zorbax Preparative SB-C8 (PrepHT Zorbax SB-C8 21.2 × 250 mm, 7 µm).Solvents selected were H 2 O (Solvent A) and CH 3 CN (Solvent B), both acidified with 0.1% trifluoroacetic acid (TFA) to increase the resolution of the peaks.The flow rate was set at 20 mL/min, and the wavelengths selected for DAD detection were 210 nm and 256 nm.The gradient of the method proceeded as follows: 1 min at 5%B (t = 1 min), 35 min from 5%B to 100%B (t = 36 min), 7 min at 100%B (t = 43 min), 2 min to initial conditions (t = 45 min).The main peaks observed on the generated chromatogram were collected and analyzed through both HPLC-UV-ESI-TOF and HPLC-UV-LRMS to dereplicate and verify purity.

Purification of Compounds
Fractions containing either more than one compound or a compound with impurities were submitted to semipreparative HPLC using the same conditions as reported for the fractionation of the crude extracts (Section 2.5).The differences rely on the flow rate (3.6 mL/min for semipreparative purification) and the gradient, which was different for each specific case.Gradients were built as follows: 1 min at chosen initial conditions, 35 min from initial %B to final %B, 7 min washing at 100%B and 2 min to restore initial conditions.A gradient annotated as 5-100 B refers to 5 as initial %B and 100 as final %B. 8 -Hydroxyfumarprotocetraric acid was purified by 20-40 B gradient, divaricatic acid purification was performed using a 45-75 B gradient, stenosporic acid was purified by 50-100 B gradient; 4 -O-Methylpaludosic acid and sekikaic were obtained purely from the crude extract fractionation.
The compounds purified from the raw lichen extracts were analyzed by HPLC-UV-LRMS, HPLC-UV-ESI-TOF and 1D/2D-NMR to confirm their identity.A compound was considered pure when both the area of the peak on the UV trace at 210 nm represented 90% of all the observed peaks, and no relevant impurities were observable in its 1 H NMR spectrum.
Liquid-based assay was performed by incubating samples (lichen extracts or isolated compounds) with conidia solutions (1 × 10 6 conidia/mL) of target microorganisms during a specific incubation time for each fungal strain (24-120 h) at 25 • C, using RPMI modified medium.The antifungal activities were scored using absorbance differences at 600 nm, between the final and the initial incubation times, as indicator of the mycelia development and resazurin as an oxidation-reduction indicator of cell viability [31].
Lichen extracts were tested by dissolving them in 100% DMSO at 2 mg/mL.Pure compounds were tested in two-fold dilutions (10 points), starting at 160 µg/mL dissolved in 100% DMSO, and 2 µL of each point was dispensed in each assay plate per triplicate.IC 50 values were calculated based on the dose-response curves obtained from both readouts, corresponding to the dose/concentration that induced half-maximum responses, as indicative of potency measure.The results obtained were analyzed using Genedata Screener software (Genedata Inc., Basel, Switzerland) [31].

Antifungal Activity of Lichen Extracts
Characterization of the antifungal properties of the six lichen extracts (A.placodiformis, C. foliacea, D. ocellatus, S. lacunose, S. lentigera and X. pokornyi) was carried out, testing them for fungal growth inhibition (absorbance) and cell viability (fluorescence), against a panel of seven fungal phytopathogens.After this evaluation, extracts from the lichens C. foliacea, S. lentigera and X. pokornyi showed total inhibition of Z. tritici based on absorbance and fluorescence readouts.
To identify the active metabolites responsible for Z. tritici inhibition, semipreparative HPLC fractions of extracts from the three lichens were conducted.Several active fractions against Z. tritici were selected for HPLC-MS analysis and further purification.

Identification of Main Active Peaks from the Raw Extracts
Antifungal active fractions were analyzed by HPLC-MS for the dereplication of known lichen metabolites.The dereplication of these molecules was then achieved by HPLC-UV-ESI-TOF analysis and comparison with the data available in the In-House database and commercial Chapman and Hall Dictionary of Natural Products, as reported in methods.Dereplication was based on retention time, biological source and the molecular formula (calculated from the m/z peaks).Confirmation of the identity of the non-dereplicated compounds was performed after HPLC purification by ESI-TOF and NMR Analysis (Table 2).Those compounds without reported biological activity were repurified and tested against the fungal phytopathogens panel described in the methods section.An approach combining feature-based molecular networking with bioassay-guided fractionation allowed the detection, isolation and identification of the metabolites responsible for the antifungal activity.The active crude extracts produced by maceration of raw lichenic biomass in acetone were analyzed by HPLC-MS/MS to generate a molecular network to compare samples and to investigate the chemical diversity by dereplication of known compounds.The main clusters of the molecular network obtained for the active lichenic extracts from the Tabernas Desert are shown in Figure 2, in which each node represents a parent ion of an MS/MS spectrum.According to the legend, the color code refers to the different super-classes of metabolites present in the cluster.The occurrence of big clusters associated with phenylpropanoids and polyketides indicates the possible presence of depsides and depsidones, characteristic molecules previously reported from lichens (Figures 3-5).

Structural Elucidation of 8 -Hydroxyfumarprotocetraric Acid
8 -Hydroxyfumarprotocetraric acid (Figure 6) was obtained as a white amorphous solid from the acetone extract of C. foliacea.Its molecular formula, C 22 H 18 O 12, was deduced from its (+)-ESI-TOF analysis ([M + NH 4 ] + : 492.1135, ∆ −0.2 ppm).The 13 C NMR spectrum of the compound (Table 3 H9) ppm.The fumaric acid moiety was confirmed by the HMBC correlation between H2 with olefinic C3 and two carboxylic carbons C1 and C4 , as well as cross-peaks between H3 with olefinic C2 , and carboxylic C1 and C4 .The connection between the dicarboxylic acid moiety with the depsidone core is evidenced by the HMBC signals of hydroxylated sp 3 methylene signal H8 with carboxylic C1 , H8 share cross-peaks with C2 , C3 and C4 aromatic sp 2 carbons.Additionally, the methyl singlet H9 showed correlations with C4 , C5 , C6 and C7 , leading to the identification of one of the phenolic acid rings.On the other hand, a cross-peak of the singlet aromatic H5 with the sp 2 aromatic carbons C3, C4 and C6, and with sp 3 C8 and C9, together with the correlation of methyl singlet H9 with aromatic C1, C5, C6 and carboxylic C7 and methylene singlet H8 cross-peaks with aromatic C3, C4 and C5, explained the configuration of the second phenolic ring.These NMR data share similarities with fumaroprotocetratic spectroscopic data published [32].showed correlations with C4′, C5′, C6′ and C7′, leading to the identification of one of the phenolic acid rings.On the other hand, a cross-peak of the singlet aromatic H5 with the sp 2 aromatic carbons C3, C4 and C6, and with sp 3 C8 and C9, together with the correlation of methyl singlet H9 with aromatic C1, C5, C6 and carboxylic C7 and methylene singlet H8 cross-peaks with aromatic C3, C4 and C5, explained the configuration of the second phenolic ring.These NMR data share similarities with fumaroprotocetratic spectroscopic data published [32].
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Anti-Phytopathogenic Activity Characterization of Pure Compounds
A total of five pure compounds (Figure 7) were obtained from the active HPLC fra tions: (1) 8-hydroxyfumarprotocetraric acid from C. foliacea (2.3 mg); (2) divaricatic ac (14.7 mg); (3) stenosporic acid from X. pokornyi (3 mg); (4) sekikaic acid (1.2 mg) and 4′-O-methylpaludosic acid from S. lentigera (10.5 mg).All pure compounds were test against the panel of fungal phytopathogens based on absorbance and fluorescence in vi assays, and their corresponding IC50 values in µg/mL were calculated (Table 4) from th corresponding dose-response curves (Figures S26-S39).The most potent compound was stenosporic acid from X. pokornyi, which presented an IC 50 against Z. tritici of 7.53 µg/mL (18.1 µM) in the absorbance-based assay and 6.59 µg/mL (15.84 µM) in the fluorescence-based assay.This compound also displayed significant antifungal activity against B. cinerea, C. acutatum, M. grisea and V. dahliae but less potency against Fusarium spp.Divaricatic acid also presented similar IC 50 values for Z. tritici (12.4 µg/mL (31.9 µM) in the absorbance-based assay and 10.67 µg/mL (27.49µM) in the fluorescence-based assay) and a similar antifungal activity pattern for the rest of the six fungal phytopathogens (Table 4). 4 -O-methylpaludosic acid only showed antifungal activity against Z. tritici, with an IC 50 of 38.87 µg/mL (87.1 µM) in the absorbance-based assay and 24.88 µg/mL (55.52 µM) in the fluorescence-based assay.8-hydroxyfumarprotocetraric acid and sekikaic acid did not display any antifungal activities against the seven fungal pathogens at the highest concentration tested of 160 µg/mL.Stenosporic acid was described in 1970 as a new depside from the lichen Ramalina stenospora collected in Florida, USA [33].Later, antimicrobial screening using extracts from X. pokornyi reported antimicrobial and antiyeast activity of stenosporic acid, but no antifungal activity has been reported for this compound against filamentous fungi, including Fusarium spp.[34].Results using our in vitro HTS platform have allowed us to describe its antifungal activity, including two Fusarium spp.
Divaricatic acid has been reported in extracts obtained from several lichen genera, including Cladonia, Flavocetraria, Parmeliopsis, Ramalia, Roccella Lecanora, Ophioparma and others, to have antibacterial, antifungal and antiparasitic activities [25].The reported MIC for this compound against B. cinerea and F. oxysporum was 6.26-12.5 mg/mL [35].Our screening results confirmed the antifungal properties of divaricatic acid but revealed a higher potency of this compound to inhibit the seven phytopathogens tested, with IC 50 values in the micromolar range, even against the extremely resistant fungus Fusarium oxysporum f. sp.cubense Tropical Race 4 (TR4).
The presence of 4 -O-methylpaludosic has been reported in extracts from several Ramalia species [36], but no biological activity of this compound has been reported to date.This molecule only showed antifungal activity against the wheat pathogen Z. tritici with IC 50 values in the micromolar range, highlighting its potential use as a specific antifungal compound to control the causal agent of S. tritici blotch.

Antitumoral Activity of Pure Compounds
As mentioned above, the five pure compounds obtained were assayed against five human cancer cell lines in an absorbance-based MTT assay, and ED 50 values were obtained.
As can be observed in Table 5, none of the compounds had significant activity against the cell lines tested.Divaricatic acid was the compound with more activity against all cell lines, except for MIA PaCa-2, reaching an ED 50 value of 15.12 µg/mL (38.93 µM) against the MCF7 breast cancer cell line.

Conclusions
Ten compounds from the bioactive lichens Cladonia foliacea, Squamarina lentigera and Xanthoparmelia pokornyi were identified by MS/MS dereplication, molecular networking and NMR experiments, nine of which were already known molecules and one of which was a new derivative of fumaroprotocetraric acid.
Stenosporic and divaricatic acids, isolated from the xerophytic lichen X. pokornyi, displayed a broad antifungal spectrum against seven relevant fungal phytopathogens, including the extremely resistant fungus F. oxysporum f. sp.cubense Tropical Race 4 (TR4).Furthermore, divaricatic acid also displayed some activity against a panel of human cancer cell lines, although this was not relevant considering that the activity was only present at high concentrations.Therefore, both compounds could represent promising broadspectrum disease control agents.Moreover, this is the first report on the biological activity of 4 -O-methylpaludosic acid isolated from S. lentigera, which exhibited specific antifungal activity against the wheat pathogen Z. tritici, the causal agent of S. tritici blotch.These results confirmed our initial hypothesis that Xerophytic lichens from gypsiferous outcrops are an important source of metabolites with anti-phytopathogenic activity.

Figure 2 .
Figure 2. Main clusters from the molecular network of lichen crude extracts.

Figure 2 .
Figure 2. Main clusters from the molecular network of lichen crude extracts.

Figure 3 .
Figure 3. Cluster 1 of the depsides family and usnic acid.Figure 3. Cluster 1 of the depsides family and usnic acid.

Figure 3 .
Figure 3. Cluster 1 of the depsides family and usnic acid.Figure 3. Cluster 1 of the depsides family and usnic acid.

Figure 7 .
Figure 7. Structure of five pure compounds tested against fungal phytopathogens.

Figure 7 .
Figure 7. Structure of five pure compounds tested against fungal phytopathogens.

Table 4 .
IC50 values in µg/mL for five pure compounds, obtained from xerophytic lichens, agai seven important fungal phytopathogens based on absorbance (Abs) and fluorescence (Flu) in vi assays.

Table 4 .
IC 50 values in µg/mL for five pure compounds, obtained from xerophytic lichens, against seven important fungal phytopathogens based on absorbance (Abs) and fluorescence (Flu) in vitro assays.

Table 5 .
ED 50 values in µg/mL for five pure compounds obtained from xerophytic lichens against five human cancer cell lines.