Astrocaryum sciophilum. The fungi were cultured, extracted with ethyl acetate, and screened for their effects on the Wnt pathway and cell proliferation. The endophytic strain Lasiodiplodia venezuelensis was prioritized for scaled-up fractionation based on its selective activity. Application of geometric transfer from analytical HPLC conditions to semi-preparative scale and use of dry load sample introduction enabled the isolation of 15 pure compounds in a single step. Among the molecules identified, five are original natural products described for the first time, and six are new to this species. An active fraction obtained by semi-preparative HPLC was re-purified by UHPLC-PDA using a 1.7 µm phenyl column. 75 injections of 8 µg were necessary to obtain sufficient amounts of each compound for structure elucidation and bioassays. Using this original approach, in addition to the two major compounds, a third minor compound identified as (R)-(-)-5-hydroxymellein (18) was obtained, which was found to be responsible for the significant Wnt inhibition activity recorded. Further studies of this compound and its structural analogs showed that only 18 acts in a highly specific manner, with no acute cytotoxicity. This compound is notably selective for upstream components of the Wnt pathway and is able to inhibit the proliferation of three triple negative breast cancer cell lines. In addition to the discovery of Wnt inhibitors of interest, this study contributes to better characterize the biosynthetic potential of L. venezuelensis.The Wnt signaling pathway controls multiple events during embryonic development of multicellular animals and is carcinogenic when aberrantly activated in adults. Breast cancers are dependent on Wnt pathway overactivation mostly through dysregulation of pathway component protein expression, which necessitates the search for therapeutically relevant compounds targeting them. Highly diverse microorganisms as endophytes represent an underexplored field in the therapeutic natural products research. In the present work, the objective was to explore the chemical diversity and presence of selective Wnt inhibitors within a unique collection of fungi isolated as foliar endophytes from the long-lived tropical palm  The ever-increasing threat of cancer is one of the greatest challenges of modern medicine . The curTaxus brevifolia, was also produced by endophytic fungi such as Taxomyces andreanae, numerous endophytes have been found to be producers of potent anticancer agents  . This acHowever, direct targeting of the Wnt pathway by acting on its most widespread components has detrimental effects on the Wnt-dependent healthy tissues resulting in poorly manageable adverse effects and failure to advance in clinical trials . More seIn the search for new natural products targeting the Wnt pathway, we sought to investigate cultivated communities of endophytic fungi in model plants known for their long-life cycle and possibly hosting steady microbial communities over many years.Astrocaryum sciophilum (Miq.) Pulle. A. sciophilum is a solitary, understory palm endemic to the northeast region of the Amazon basin   . Its remuriname) . This suuriname) . Moreoveuriname) .A. sciophilum were cultured, extracted, and screened for their effects on cell proliferation and Wnt pathway inhibition. Among them, the endophytic strain Lasiodiplodia venezuelensis Burgess, Barber, and Mohali . The extracts were then partitioned with water (W). All extracts were screened for their effects on Wnt pathway inhibition. The specificity was controlled by analyzing general cytotoxicity. As a system to screen compounds for the potential anti-Wnt signaling activities, we used the BT-20 TNBC cells, stably transfected with the TopFlash reporter construct, responding by luciferase expression to stimulation with purified Wnt3a  and low cytotoxicity (Renilla inhibition IC50 > 500 μg/ml (estimated)). Based on the bioactivity results and a preliminary metabolite profiling revealing an interesting composition , the A02 strain was prioritized and cultivated on a large scale (100 14 cm diameter Petri dishes) and extracted with the same procedure as for the screening. The large-scale EtOAc extract (A02Et) was then tested in an independent series of measurements and confirmed the anti-Wnt activity observed on a small scale .A collection of 15 strains isolated as cultivable endophytes from the leaves of ed Wnt3a . EtOAc a2O (7:3). This process yielded an apolar hexane fraction (A02EtH) and an enriched hydroalcoholic fraction (A02EtM) .A first chemical profile of the bioactive A02Et extract by HPLC-PDA-ELSD revealed the presence of a significant amount of lipophilic compounds as shown by ELSD. In order to rule out a possible interference of lipid compounds in the Wnt assay, the EtOAc extract was further simplified by liquid/liquid partitioning hexane/methanol:H(A02EtM) . The spe50 was 15 ± 3.9 μg/ml, and cytotoxic IC50 was >200 μg/ml.For efficient isolation of the active compounds, the reverse phase HPLC-PDA-ELSD conditions were optimized in order to obtain an efficient separation . Those cDeep Metabolome Analysis).The A02EtM and the F19 fraction were profiled by UHPLC-PDA-HRMS/MS using data-dependent acquisition mode followed by metabolite annotation to estimate its chemical composition. The detailed procedure for metabolite annotation is described in the following section , corresponding to 4 degrees of unsaturation. In MS1 and MS/MS, this ion was dereplicated as decumbic acid, already isolated from Lasiodiplodia theobromae , corresponding to 6 degrees of unsaturation. In MS1, this ion was dereplicated as 3,4-dihydro-4,8-dihydroxy-3-methyl-1H-2-benzopyran-1-one (4-hydroxymellein), also isolated from Lasiodiplodia theobromae , annotated the compound corresponding to this feature as 3,4-dihydro-3,8-dihydroxy-3-methyl-1H-2-benzopyran-1-one, which differed in the hydroxylation pattern. This indicated that this feature was an hydroxymellein derivative, but its exact structure could not be asserted at this level.A careful analysis of the HRMS and HRMS/MS spectra of the active fraction F19 in the chemical profile of the extract revealed that it contained molecular ions corresponding to two distinct molecules . The fireobromae . The seceobromae . MS/MS dH 6.32 and 6.00 (J = 2.8 Hz) corresponding to an exocyclic double bond that replaced the methyl at δH 2.13 of the decumbic acid (11), and an additional methine at δH 3.72  revealed that an isomer of decumbic acid with a close structure was also present (10). The minor signals in this fraction seem to correspond partly to the hydroxymellein derivative (18) dereplicated by MS/MS, but a full de novo identification could not be made at this stage /1.3 (50%)/ 0.4 (10%) moles. The main signals in the NMR spectrum corresponded to decumbic acid, as suggested by the MS/MS annotation. However, signals at δis stage .In order to identify which metabolite(s) was responsible for the Wnt inhibition, careful purification was needed since none of them was described as an anti-Wnt inhibitor.Several analytical HPLC conditions were tested but did not enable a baseline separation of the three constituents. Further UHPLC-PDA profiling of this fraction using an analytical phenyl column packed with 1.7 µm silica beads finally yielded an efficient separation of the three constituents . This chSince the loading capacity of the UHPLC column was very limited, a series of 75 injections of 8 µg each was performed . All sepS,4R)-3-carboxy-2-methylene-heptan-4-olide (10), a toxin responsible for the decay of tropical fruit, and its inactive derivative, decumbic acid (11), isolated from L. theobromae -(-)-5-hydroxymellein (18). Melleins are a subgroup of 3,4-dihydrocoumarins that belong to the subgroup of isocoumarins, a well-known polyketides family. Such derivatives are produced by bacteria, fungi, higher plants, insects, lichens, and marine sponges, the most important source of mellein being the fungi. They have shown a wide range of biological activities (R)-Mellein  and -4-hydroxymellein and (3R)-5-hydroxymellein have already been isolated from Lasiodiplodia sp. 5-Hydroxymellein has been already isolated from Lasiodiplodia theobromae but never reported in L. venezuelensis -(R)uelensis . It has ll lines . The NMRR)-(-)-5-hydroxymellein (18) was responsible for the significant Wnt inhibition activity of the fraction F19, with a Wnt IC50 of 6.7 ± 1.2 μg/ml (58 ± 16 µM) (cytotoxic IC50 > 20 μg/ml (>500 µM)) ) .L. venezuelensis. Moreover, the metabolome of this particular species was not described in the literature. In order to document the composition of this strain and understand its chemodiversity, we took advantage of having the UHPLC-HRMS/MS metabolite profiling and the NMR analysis of the high-resolution semi-preparative HPLC fractions to combine this information for a deep metabolome characterization. Furthermore, we highlighted a cluster of potential analogs of the active 5-hydroxymellein 18 and exploited the possibility of isolating them to further assess any structure-activity relationship .The first chemical profiling of the extract by UHPLC-HRMS/MS indicated a potentially interesting chemical composition of the ethyl acetate extract of de novo structure identification is described hereafter.From the 33 fractions obtained from semi-preparative HPLC semi-preparative purification, fifteen pure compounds (1–15) were successfully isolated in a single step . Ten com19H15NO4 with a m/z of 214.1071 [M + H]+ , which corresponded to 4 degrees of unsaturation. Analysis of the 1H, 13C and HSQC NMR data of 1 indicated the presence of a dihydropyran scaffold consisting of two olefinic carbons at δC 135.0 (C-3) and 123.3 (C-4) and three oximethines at δC 80.6 (C-2), 71.5 (C-5), 66.2 (C-6). In addition to these signals, a methyl group at δH 1.19 , an oximethine at δH 3.71 , two olefinic protons at δH 7.00  and δH 6.18 , and an ester carbonyl at δC 165.4 (C-3') were observed. Sequential COSY correlations from H-6 at δH 4.17  to H-5 (δH 4.74), H-4 (δH 6.10), and H-3 (δH 6.32) and HMBC correlations from the olefinic proton H-4 at δH 6.10  to C-2 and C-6 and from the oximethine proton H-6 to C-5 and C-2 allowed defining a 3,6-dihydro-2H-pyran-3-ol ring. The COSY correlations from H-1' to H-2' and H-6 and HMBC correlations from H-1' and H-2' to the ester carbonyl C-3' positioned a propenamide side chain at C-6. The 9.8 Hz coupling constant of the set of olefinic protons H-1' and H-2' demonstrated the cis configuration of the double bond on this side chain. Correlation from the methyl protons H-8 to the oximethine C-7 (δC 69.9) and C-2 located a 1-hydroxyethyl group in C-2. Further, COSY correlations from H-7 to H-8 and H-2 supported the position of this second side chain. The ROESY correlations from H-6 to H-5 and H-2 and the small coupling constant between H-5 and H-6 (J = 2.8 Hz) indicated the relative configuration of the 3,6-dihydro-2H-pyran-3-ol ring  configurations. To define the absolute configuration of C-7, the S-MTPA and R-MTPA ester derivatives were synthesized from the (R)- and (S)-α-methoxy-α-trifluoromethylphenylacetyl chloride, respectively (R-MTPA ester relative to the S-MTPA ester were consistent with a C-7 (R) configuration (Z)-3[-2-hydroxy-5[(1R)-1-hydroxyethyl]cyclohex-3-en-1-yl]prop-2-enamide.Compound 1 (F6) was isolated as an oil. The ESI(+)-HRMS experiment indicated a molecular formula of C-ol ring . To esta-ol ring . After tectively  , which corresponded to 5 degrees of unsaturation. Analysis of the 1H-NMR and HSQC data of 3, as this was the case for 1 indicated the presence of a dihydropyranone scaffold consisting of two olefinic carbons at δC 121.1 (C-3) and 146.1 (C-4) two oximethines carbons at δC 60.8 (C-5) and 76.1 (C-6). In addition to these signals, two olefinic protons at δH 5.90  and δH 5.71 , two oximethines protons at δH 5.29  and at δH 3.77 , two methyl groups at δH 1.05  and δH 2.01 , and two protons from hydroxy groups at δH 5.60  and at δH 4.87 . Analysis of the HMBC spectrum revealed correlations from the olefinic proton H-3 to the ester carbonyl C-2 at δC 162.9 and to C-5 and from the second olefinic proton H-4 to C-5, C-6, and C-2. Furthermore, COSY correlations between the olefinic protons H-3 and H-4 and from H-4 to H-5 defined the dihydropyranone moiety. COSY correlation from H-5 to the hydroxyl group at δH 5.60 placed the first hydroxy group in position C-5. Sequential COSY correlations from H-6 to H-1', H-2', and H-3' and HMBC correlations from the methyl H-5' to C-4' (δC 66.8) and C-3' (δC 73.4) positioned the side chain at C-6. COSY correlation between H-4' and the hydroxyl group at δH 4.87 placed the second hydroxy group in position C-4'. HMBC correlations from the ester carbonyl at δC 169.5 to H-3' and to the methyl at δH 2.01 positioned an acetate in C-3'. The 11.1 Hz coupling constant of the set of olefinic protons H-1' and H-2' demonstrated the cis configuration of the double bond on the side chain. The ROESY correlation between H-5 and H-6 and the J5,6 value of 2.0 Hz positioned the propenamide side chain at C-6 in a pseudo-equatorial orientation and the hydroxy group at C-5 in axial orientation. Moreover, the positive Cotton effect in the circular dichroism curve (Δε 260–290 nm) was correlated with the C-6(S) configuration as described for substituted dihydro-α-pyrones in the literature  and the ROESY correlations from H-3' to H-4' and OH4’ and from H-4' to H-2' indicated a 3'S,4'R or 3'R,4'S configuration of the side chain (Compound 3 (F8) was isolated as an oil. The ESI(+)-HRMS experiment indicated a molecular formula of Cterature . The smade chain .Z,3S,4R)‐4‐hydroxy‐1‐pent‐1‐en‐3‐yl acetate.To establish the absolute configuration of 3, the ECD spectrum was calculated based on the relative configuration proposed by NMR and compared to the experimental data . After tHyptis pectinata , which corresponded to 4 degrees of unsaturation. Analysis of the 1H and 13C-DEPTQ NMR data of 4 showed the presence of two methyl groups at δC 15.6 (C-10) and 21.2 (C-12); 4 oximethines carbons at δC 63.4 (C-4), 70.7 (C-8), 74.4 (C-9), and 78.3 (C-5); 4 olefinic carbons at δC 122.8 (C-2), 127–7 (C-6), 135.4 (C-7), and 147.1 (C-3); two ester carbonyls at δC 165.9 (C-1) and 172.6 (C-11). A n-nonane chain was easily followed through the COSY correlations from the ethylenic proton H-2 to the methyl H-10. The HMBC correlation of the carbonyl C-1 with the olefinic set of protons H-2 and H-3 positioned the amide group at one side of the chain and an acetate group was placed on the other side thanks to the HMBC correlation of the carbonyl C-11 to the oximethine H-9 and to the methyl H-12. The 9.8 and 11.3 Hz coupling constants for the sets of olefinic protons H-2/H-3 and H-6/H-7, respectively, demonstrated the cis configuration of both double bonds -10-amino-3,6,7-trihydroxy-10-oxodeca-4,8-dien-2-yl-acetate.Compound 4 (F9) was isolated as an oil. The ESI(+)-HRMS experiment indicated a molecular formula of Cle bonds . Moreove10H14O4 with a m/z of 199.0964 [M + H]+ , which corresponded to 4 degrees of unsaturation. Analysis of the 1H and edited HSQC NMR data indicated the presence of two compounds, the minor one being  or -4,5-dihydroxymellein. The major compound  was linked to the following NMR signals: one methyl group at δC 18.0 (C-10), six olefinic carbons at δC 120.8 (C-2), 139.6 (C-3), 127.4 (C-4), 139.0 (C-5), 131.1 (C-8), and 129.5 (C-9), and two oximethines carbons at δC 71.5 (C-6) and 76.9 (C-7). Sequential COSY correlation from the methyl H-10 at δH 1.67  to H-9 (δH 5.72), H-8 (δH 5.45), H-7 (δH 3.92), H-6 (δH 4.45), and H-5 (δH 5.72) defined one side of the molecule. HMBC correlations from H-7 to C-5, C-6, and C-9 and from the methyl H-10 to C-9 and C-8 confirmed this assignment. The HMBC correlation of the carbonyl at δC 170.0 (C-1) with the olefinic proton H-3 and the COSY correlation of H-3 with an olefinic proton H-2 at δH 5.74 positioned the carboxylic acid group in C-1. HMBC correlation of H-3 with C-5 and COSY correlation of H-3 with the olefinic proton H-4 at δH 7.32 and of H-4 and H-5 helped position the olefinic groups and link both chains. The 11.5 Hz coupling constant for the set of olefinic protons H-2/H-3 and H-4/H-5 demonstrated the cis configuration of those double bonds. The 15.2 Hz coupling constant for the third set of olefinic protons H-8/H-9 demonstrated the trans configuration of the double bond -6,7-dihydroxydeca-2,4,8-trienoic acid.Compound 6 (F11) was isolated as an oil. The ESI(+)-HRMS experiment indicated a molecular formula of Cble bond . CompounEdenia gomezpompae, respectively. Compounds 15 and 16 were thus identified as the new naphthoquinone spiroketals enantiomers (+)-(R)-CJ-12,372 and (+)-(R)-palmarumycin EG1  and a m/z of 333.1253 [M−H]− , which corresponded to 13 degrees of unsaturation. 1H and 13C NMR spectra suggested that the molecule was a deoxypreussomerin derivative related to palmarumycin CP19  and the substitution pattern in the non-aromatic ring  were identical in both molecules. However, the signals from the third aromatic ring were different, and the multiplicity  indicated a 1,2,4-trisubstituted aromatic ring instead of a 1,2,3-trisubstituted aromatic ring in palmarumycin CP19. 17 has a single stereogenic center. Its absolute C-1 configuration was deduced by comparing the sign of the optical rotation with the one of the closely related palmarumycin CP19 (S)-palmarumycin CP19 is negative and 17 has a positive optical rotation, the C-1 configuration was thus determined as R (R)-4-methoxy-3,4-dihydro-2H-spirodioxin]-6-ol.Compound 17 (F32) was isolated as an oil. The ESI(-)-HRMS experiment indicated a molecular formula of Ccin CP19 . The sigcin CP19 . Since tned as R . CompounLasiodiplodia pseudotheobromae  (In order to obtain a comprehensive survey of the metabolome of ork (MN) . The resin silico fragmentation spectra of all the compounds present in the DNP (DNP-ISDB)  (Annotation was performed against an in-house database containing the NP-ISDB) . Spectrariaceae) . The topriaceae) . For allriaceae) . The annriaceae) . The fulPie charts on the nodes and their proportions refer to the relative intensities of the ions in the extract  or in the fractions , based on the respective areas of the corresponding extracted ion chromatography (XIC). The numbering of the clusters corresponds to the component index number.In order to have a confident annotation of the largest possible number of metabolites, all nodes corresponding to the protonated molecule of the 18 isolated compounds were located in the MN  and occu7 (F13), 13 (F22), 18 (F19)) and C_13 (8 (F14), 9 (F16)), . All these nodes were thus unambiguously identified.In a first instance, the MN was analyzed to assess the presence of potential analogs of the active compound 18. For this, two clusters containing the nodes of all isolated mellein derivatives were analyzed in-depth to evidence additional putative isocoumarin analogs: cluster C_54 (Lasiosiplodia) were carefully analyzed. This enabled annotating with good confidence eight additional nodes (n2 and n8) as isocoumarin derivatives , 9 (F16), and n11 (F16). All these compounds are 4-hydroxy derivatives without hydroxyl in position 5, while 18 did not have any hydroxylation in 4. However, the ICions see . The ann50 measurement of isolated compounds  confirmed that these compounds did not show any specific inhibition of Wnt3a-induced signal up until estimated concentrations of ∼500 μM, indicating that 18 acts in a highly specific manner   . Anotheran-2-one .Compounds 2, 6, and 14 were spotted in three different clusters of fatty acyl derivatives , was isolated, but its associated feature could not be evidenced in the MN in positive mode since it ionizes in negative mode only  derivatives  was in confrontation with another wood-decaying fungus Eutypa lata and exhibited toxicity towards other fungi  using the residual CD3OD  or DMSO-d6  as internal standards for 1H and 13C NMR, respectively, and coupling constants (J) are reported in Hz. Complete assignments were obtained based on 2D-NMR experiments: COSY, ROESY, edited HSQC, and HMBC. HRESIMS data were obtained on a Q-Exactive Focus mass spectrometer , using heated electrospray ionization (HESI-II) source. Analytical HPLC was carried out on a HP 1260 system equipped with a photodiode array and an ELSD detector . Semi-preparative HPLC-UV analyses were conducted on a Shimadzu system  equipped with LC-20A module pumps, a SPD-20A UV/VIS, a 7725I Rheodyne® valve, and a FRC-10A fraction collector. The system is controlled by the software LabSolutions from Shimadzu.NMR spectroscopic data were recorded on a Bruker Avance Neo 600 MHz NMR spectrometer equipped with a QCI 5 mm Cryoprobe and a SampleJet automated sample changer . Chemical shifts are reported in parts per million (The spectra obtained by electronic circular dichroism (ECD) and UV were analyzed on a J-815 circular dichroism spectrometer . The solvent used was methanol. The cell temperature was 24.5°C and stabilized using a thermostat ED circulation bath  and a PFD-350S thermostat (Jasco). The set scan speed was 200 nm/min in continuous mode with a scan start at 800 nm and an end at 200 nm. The optical rotations were measured in methanol solutions on Jasco P-1030 polarimeter in a 1 cm tube.Astrocaryum sciophilum palm tree leaves were sampled in August 2017 at the Nouragues ecological research station in French Guiana. Leaf samples were collected at the Inselberg research station (4°05'N–52°41'W) by randomly cutting 5 × 3 cm pieces of fresh leaflets and rachis from individual plants using sterile tweezers. The pieces were placed in Eppendorf tubes containing Potato Dextrose Broth 1/4 (PDB) medium to allow regrowth of the fungi and tubes containing cetyltrimethylammonium bromide (CTAB) to preserve the genetic material. Petri dishes containing “potato dextrose agar” medium and aureomycin were also left open on the stipe in order to sample fungi from the environment close to the palm tree. These media were enriched with aureomycin to avoid the growth of bacteria and promote the growth of fungi only.2), placed individually in Petri dishes containing potato dextrose agar medium , amended with aureomycin (25 ppm. L−1) in 9 cm Petri dishes, and cultured at room temperature. Each individual emerging hypha fragment was removed and placed in 9 cm Petri dishes containing PDA and aureomycin. The isolated and selected strains were then individually cultured on a small scale in 9 cm Petri dishes containing PDA medium without aureomycin. Each strain has been integrated into the dynamic fungal library of Agroscope, whose content is available on the web (database Mycoscope: www.mycoscope.ch), in vials containing 5 ml of diluted PDB  aqueous solution (1:4) at 4°C.The surface of the plant fragments was thoroughly washed for 3 hours under a stream of tap water in a suitable container and then washed in three successive baths of sterile water for 10 min each under a laminar flow hood. This non-chemical surface cleaning method was used to reduce the possible loss of diversity due to aggressive sterilization using alcoholic solutions or even Paraquat . The surhttps://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify the strain. The specimens were then entered in the dynamic mycotheca of Agroscope and registered in the fungal database (www.mycoscope.ch) under the reference numbers 1883 to 2024.Identification of the isolated strains was performed by extraction of the ribosomal DNA (phenol/chloroform), PCR amplification of the ITS1F and ITS4 region, and sequencing . The obtained sequences were then submitted to BLAST on NCBI  in 10 cm Petri dishes containing PDA culture medium. The culture medium was then extracted by maceration in ethyl acetate (EtOAc) for 24 h at room temperature under agitation. The organic phase was recovered by vacuum filtration and washed three times with Millipore Corporation (MilliQ) water. The organic and water phases were dried under reduced pressure with a rotary evaporator to yield crude mixtures. Agar from uninoculated PDA plates was used as controls.Lasiodiplodia venezuelensis strain was conducted on 100 14 cm Petri dishes to yield the factions A02Et (1 g) and A02W (335 mg). 600 mg of the fraction A02Et was then degreased to yield fractions A02EtH (320 mg) and A02EtM (250 mg).Strains were cultivated for 15 days at room temperature in 14 cm Petri dishes containing PDA medium. Culture media were then consecutively extracted three times by maceration in EtOAc for 24 h at room temperature under agitation . The combined organic solution was washed three times with MilliQ water. The organic and water phases were dried under reduced pressure with a rotary evaporator to yield the ethyl acetate fraction (Et) and the water fraction (W). The Et fraction was then degreased by liquid/liquid partition in a hexane/methanol:water (1/1) system with methanol:water (7:3) to give the hexane (EtH) and the methanol/water fraction (EtM) . Large-sμL/min and the injection volume was set at 2 μL. The optimized HESI-II parameters were as follows: source voltage, 3.5 kV (pos), 4kV (neg); sheath gas flow rate (N2), 55 units; auxiliary gas flow rate, 15 units; spare gas flow rate, 3.0; capillary temperature, 275°C (pos), 320°C (neg); S-Lens RF Level, 45. The data-dependent MS/MS events were performed on the four most intense ions detected in full-scan MS (top three experiments). The MS/MS isolation window width was 1 Da, and the normalized collision energy was set to 35 units. In data-dependent MS/MS experiments, full scans were acquired at a resolution of 35,000 FWHM (at 200 m/z) and MS/MS scans at 17,500 FWHM, both with a maximum injection time of 50 ms. After being acquired in a MS/MS scan, parent ions were placed in a dynamic exclusion list for 2.0. In positive mode, the di-isoctyl phthalate C24H38O4 [M + H]+ ion (m/z 391.28429) was used as an internal lock mass. The mass analyzer was calibrated using a mixture of caffeine, methionine-arginine-alanine-acetate (MRFA), sodium dodecyl sulfate, sodium taurocholate, and Ultramark 1,621 in an acetonitrile/methanol/water solution containing 1% formic acid by direct injection. An Acquity UPLC photodiode array detector was used to acquire UV spectra detected in the 200–500 nm range.Metabolite profiling of the extracts was performed by UHPLC-PDA-CAD-HRMS/MS as follows. The separation was achieved on an Acquity BEH C18 column . The temperatures in the autosampler and in the column oven were, respectively, set at 10 and 40°C. The mobile phase constituted of water (A) and acetonitrile (B), both containing 0.1% formic acid; the separation was performed with a linear gradient from 5 to 100% of B in 7 min followed by a 1 min isocratic step at 100% of B. The flow rate was set at 600 4. The ADAP chromatogram builder was employed with a minimum height of 5 E4, a minimum group size of scans of 5, a minimum group intensity threshold of 5 E4, a minimum highest intensity of 5 E4, and a m/z tolerance of 8 ppm. The wavelets ADAP algorithm was used for chromatogram deconvolution with the following settings: a single-to-noise (S/N) threshold of 10, intensity window SN, a minimum feature height of 5 E4, a coefficient area threshold of 100, a peak duration range between 0.02 and 0.9 min, and a wavelet range between 0.00 and 0.05 min. The m/z and retention time (RT) ranges for MS2 scan pairing were, respectively, set to 0.025 Da and 0.1 min. The chromatograms were deisotoped using the isotope peak grouper algorithm with a m/z tolerance of 8 ppm, a RT tolerance of 0.08 min, and a maximum charge of 2, while the representative isotope used was the most intense. The peak alignment was performed using the join aligner method with a m/z tolerance of 8 ppm, a RT absolute tolerance of 0.05 min, and a weight for m/z and RT at 20. An adduct search  was performed on the peak list with an RT tolerance of 0.01 min, a m/z tolerance of 8 ppm, and a maximum relative peak height of 1,000%. A complex search was performed on the peak list with an RT tolerance of 0.01 min, a m/z tolerance of 8 ppm, and a maximum relative peak height of 1,000%. The peak list was then gap-filled using the same RT and m/z range gap filler with a m/z tolerance of 8 ppm. A custom database of the fungi group  was applied for in silico identification from the Dictionary of Natural Product (DVD version 26.2) (The raw files containing UHPLC-HRMS/MS data were converted into mzXML files using the msConvert software. The mzXML files were then processed using MZmine (v2.51) . Mass deon 26.2) .in silico MS/MS fragmentation spectra match, called “initial rank.” A chemotaxonomical filter was applied to weight the search according to the occurrence of structures in Botryosphaeriaceae to give a ranking of the top six most probable structures and was called “final rank.” The output was visualized using Cytoscape v3.7.1 (https://cytoscape.org/). Peak areas of different analyses were represented as pie chart diagrams. Node sizes were proportional to the peak areas of the extract. The full MS dataset is uploaded and accessible on the GNPS servers as Massive Data set n° MSV000086605.The MZmine files were exported in MGF format for the processing of the molecular network (MN) in the Global Natural Products Social (GNPS) platform . In orde2O (A) and MeOH (B), both containing 0.1% formic acid (FA). The column was equilibrated with 5% of B for 15 min. The separation was performed with a gradient mode from 5 to 60% of B over 70 min, then from 60 to 100% of B over 20 min. The column was then washed with 100% of B for 10 min and equilibrated with 5% of B over 15 min. The flow rate was 1 ml/min, the injection volume 20 μL, and the sample concentration 1 mg/ml in MeOH. The UV absorbance was measured at 254 and 280 nm and the ELSD set at 45°C, 3.5 bar N2, gain 8.HPLC-PDA analyses were conducted as follows: Waters® X-bridge C18 column   equipped with a Waters® C18 pre-column cartridge holder (20 × 4.6 mm i.d.); solvent system Hμm) using a mobile phase consisting of water (A) and acetonitrile (B), both with 0.1% FA. The separation was performed with a linear gradient from 5 to 100% of B in 7 min followed by a 1 min isocratic step at 100% of B. The flow rate was set at 600 μL/min and the injection volume at 2 μL. The temperatures in the autosampler and in the column oven were, respectively, set at 10 and 40°C. UV data were acquired from 200 to 600 nm and the ELSD temperature was set at 45°, the pressure at 3.5 bars,, and the gain at 8. Mass data were obtained by a fully automated acquisition at 10 Hz for m/z between 100 and 1,000. Data acquisition, instrument control, and data processing were performed with the Masslynx® software (Waters®).UHPLC-UV-ELSD-QDa analyses were performed on an Acquity BEH C18 column  and MeOH (B), both containing 0.1% FA. The column was equilibrated with 5% of B for 15 min. The separation was performed with a gradient mode from 5 to 60% of B over 71.63 min, then from 60 to 100% of B over 21.46 min. The column was then washed with 100% of B for 10 min and equilibrated with 5% of B over 15 min. The flow rate was 17 ml/min. These conditions were calculated from a geometric gradient transfer from the analytical HPLC using the software developed by Guillarme et al.  equipped with a Waters® C18 pre-column cartridge holder (10 × 4.6 mm i.d); solvent system He et al. . The UV e et al. . The A02e et al. . AccordiμL loop. Data acquisition, data handling, and instrument control were performed with Empower Software (Waters®). The separation analyses of fraction F19 were performed on an Acquity BEH phenyl column  using a mobile phase consisting of water (A) and acetonitrile (B), both with 0.1% FA. The separation was conducted with an isocratic mode: 5% of B during 15 min. The temperatures in the autosampler and column oven were, respectively, set at 10 and 40°C. UV data were acquired at 210 nm with a time constant set at 100 ms and a data sampling rate at 20 points/s. The flow rate was set at 300 μL/min. The fraction was concentrated at 4 mg/ml in ACN. 75 injections of 2 μL were performed. Three peaks were collected based on the time frame by which their UV signals were received. The collection for the first peak was set from 5.67 to 6.39 min, the second one from 7.09 to 7.91 min, and third one from 9.05 to 10.12 min. This allowed yielding 94 μg of peak 1 (compound 10), 83 μg of peak 2 (compound 11), and 30 μg of peak 3 (compound 22). The reproducibility of the 75 injections can be accessed in Separation was performed on a Waters® Acquity UPLC system  equipped with a UV-Vis programmable detector and an autosampler with a 2 max 205 (3.94), 321 (2.32) nm. 1H NMR  δ 1.19 , 3.71 , 4.04 , 4.17 , 4.74 , 6.10 , 6.18 , 6.32 , 7.00 ; 13C NMR  δ 18.8 (C-8), 66.2 (C-6), 69.9 (C-7), 71.5 (C-5), 80.6 (C-2), 123.3 (C-4), 124.8 (C-2'), 135.0 (C-3), 142.7 (C-1'), 165.4 (C-3'). ESI(+)-HRMS m/z 214.1071 [M + H]+ . For NMR spectra, see (Z)-3--3-Hydroxy-6-((R)-1-hydroxyethyl)-3,6-dihydro-2H-pyran-2-yl)acrylamide (1) (F6). max 209 (3.49) nm. 1H NMR  δ 3.81 , 3.88 , 6.91 ; 13C NMR  δ 34.0 (C-4), 53.1 (C-7), 131.7 (C-2), 139.9 (C-3), 168.2 , 173.8 (C-5). ESI(+)-HRMS m/z 189.0394 [M + H]+ . For NMR spectra, see Aconitate B (6-methyl ester trans-aconitate) (2) (F7). UV(MeOH) λmax 205 (3.99), sh 264 (3.37), 336 (3.25) nm. 1H NMR  δ 1.05 , 2.01 , 3.77 , 4.02 , 4.87 , 5.25 , 5.29 , 5.60 , 5.71 , 5.90 , 6.03 , 7.07 ; 13C NMR  δ 18.4 (C-5'), 20.7 (C-7'), 60.8 (C-5), 66.8 (C-4'), 73.4 (C-3'), 76.1 (C-6), 121.1 (C-3), 128.2 (C-1'), 129.7 (C-2'), 146.1 (C-4), 162.9 (C-2), 169.4 (C-6'). ESI(+)-HRMS m/z 257.1020 [M + H]+ . For NMR spectra, see -4-Hydroxy-1--3-hydroxy-6-oxo-3,6-dihydro-2H-pyran-2-yl)pent-1-en-3-yl acetate (3) (F8). max 206 (2.65), sh 266 (1.93), 343 (1.85) nm. 1H NMR  δ 1.24 , 2.05 , 4.18 , 4.46 , 4.90 , 5.33 , 5.78 , 5.93 , 6.09 , 7.07 ; 13C NMR  δ 15.6 (C-10), 21.2 (C-12), 63.4 (C-4), 70.7 (C-8), 74.4 (C-9), 78.3 (C-5), 122.8 (C-2), 127.7 (C-6), 135.4 (C-7), 147.1 (C-3), 165.9 (C-1), 172.6 (C-11). ESI(+)-HRMS m/z 274.1291 [M + H]+ . For NMR spectra, see -4,5,8,9-Tetrahydroxydeca-2,6-dienamide (4) (F9). max 207 (3.64), sh 240 (2.98), 303 (2.88) nm. Isomer 1: 1H NMR  δ 1.26 , 4.20 , 5.37 , 6.90 , 7.05 , 7.54 ; 13C NMR  δ 18.9 (C-2'), 68.7 (C-1'), 85.2 (C-3), 113.6 (C-8), 114.7 (C-4), 116.8 (C-6), 137.4 (C-5), 151.0 (C-9), 158.2 (C-7), 172.0 (C-1). Isomer 2: 1H NMR  δ 1.18 , 4.05 , 5.34 , 6.88 , 7.07 , 7.54 ; 13C NMR  δ 18.0 (C-2'), 69.7 (C-1'), 85.7 (C-3), 113.1 (C-8), 115.0 (C-4), 116.9 (C-6), 137.4 (C-5), 150.6 (C-9), 158.3 (C-7), 171.9 (C-1). ESI(+)-HRMS m/z 195.0652 [M + H]+ . For NMR spectra, see 3ξ-(1ξ-Hydroxyethyl)-7-hydroxy-1-isobenzofuranone (5) (F10). UV(MeOH) λmax 205 (3.61), sh 225 (3.51), 257 (3.42) nm. 1H NMR  δ 1.67 , 3.92 , 4.45 , 5.45 , 5.72 , 5.74 , 5.75 , 7.01 , 7.32 ; 13C NMR  δ 18.0 (C-10), 71.5 (C-6), 76.9 (C-7), 120.8 (C-2), 127.4 (C-4), 129.5 (C-9), 131.1 (C-8), 139.0 (C-5), 139.6 (C-3), 170.0 (C-1). ESI(+)-HRMS m/z 199.0964 [M + H]+ . For NMR spectra, see -6,7-Dihydroxydeca-2,4,8-trienoic acid (6) (F11). UV(MeOH) λmax 213 (4.01), 222 (4.02) nm. 1H NMR  δ 1.28 , 4.85 , 4.94 , 6.85 , 7.11 ; 13C NMR  δ 18.1 (C-11), 64.4 (C-4), 82.7 (C-3), 108.1 (C-9), 119.1 (C-7), 124.2 (C-10), 126.2 (C-6), 148.8 (C-5), 156.1 (C-8), 169.9 (C-1). ESI(+)-HRMS m/z 211.0601 [M + H]+ . For NMR spectra, see (+)--4,5-Dihydroxymellein (7) (F13). max 211 (3.88) nm. 1H NMR  δ 1.52 , 4.56 , 4.72 , 6.97 , 7.56 ; 13C NMR  δ 16.3 (C-11), 67.7 (C-4), 80.0 (C-3), 108.4 (C-9), 118.4 (C-7), 119.8 (C-5), 137.7 (C-6), 143.2 (C-10), 162.9 (C-8), 171.0 (C-1). ESI(+)-HRMS m/z 195.0654 [M + H]+ . For NMR spectra, see (-)--4-Hydroxymellein (8) (F14). max 209 (3.57) nm. 1H NMR  δ 1.47 , 4.56 , 6.94 , 7.08 , 7.57 ; 13C NMR  δ 18.2 (C-11), 69.5 (C-4), 81.6 (C-3), 108.0 (C-9), 117.7 (C-5), 117.8 (C-7), 137.8 (C-6), 144.1 (C-10), 162.9 (C-8), 170.2 (C-1). ESI(+)-HRMS m/z 195.0652 [M + H]+ . For NMR spectra, see (-)--4-Hydroxymellein (9) (F16). 1H NMR  δ 0.99 , 1.48 , 1.72 , 3.72 , 4.80 , 6.00 , 6.32 ; 13C NMR  δ 14.0 (7), 19.3 (6), 38.7 (5), 81.0 (4), 125.2 (8), 135.6 (2), 170.6 (1), 172.5 (9). ESI(-)-HRMS m/z 183.0661 [M-H]- . For NMR spectra, see -3-Carboxy-2-methylene-heptan-4-olide (10) (F19). 1H NMR  δ 0.96 , 1.48 , 1.59 , 2.09 , 2.13 , 5.15 ; 13C NMR  δ 10.6 (8), 14.1 (7), 19.1 (6), 35.7 (5), 83.0 (4), 136.9 (2), 151.2 (3), 165.0 (9), 175.3 (1). ESI(-)-HRMS m/z 183.0660 [M-H]- . For NMR spectra, see Decumbic acid (11) (F19). 1H NMR  δ 0.98 , 1.29 , 1.46 , 1.55 , 1.70 , 1.82 , 2.70 , 2.99 , 4.49 ; 13C NMR  δ 14.1 (C-7), 14.6 (C-8), 19.9 (C-6), 38.0 (C-5), 41.1 (C-2), 55.5 (C-3), 81.4 (C-4), 174.1 (C-9), 179.7 (C-1). ESI(-)-HRMS m/z 185.0818 [M-H]- . For NMR spectra, see Decumbic acid B (12) (F20). max 208 (3.78), 236 (3.52) nm. 1H NMR  δ 1.49 , 2.92 , 3.03 , 4.75 , 6.78 , 6.85 , 7.45 ; 13C NMR  δ 20.9 (C-11), 35.2 (C-4), 77.8 (C-3), 109.3 (C-9), 116.7 (C-7), 119.3 (C-5), 137.4 (C-6), 141.6 (C-10), 163.2 (C-8), 171.6 (C-1). ESI(+)-HRMS m/z 179.0702 [M + H]+ . For NMR spectra, see (-)-(R)-Mellein (13) (F22). 1H NMR  δ 0.91 , 1.32 , 1.35 , 1.69 , 1.86 , 3.37 , 5.60 , 6.14 ; 13C NMR  δ 14.3 (C-8), 23.5 (C-7), 31.0 (C-6), 32.1 (C-5), 49.8 (C-2), 125.4 (C-9), 142.7 (C-3), 172.0 (C-4), 178.5 (C-1). ESI(+)-HRMS m/z 187.0965 [M + H]+ . For NMR spectra, see (2R)-Butylitaconic acid (14) (F24). 1H NMR  δ 1.89 , 2.05 , 2.10 , 2.35 , 5.10 , 6.77 , 6.85 , 6.94 , 6.98 , 7.46 , 7.47 , 7.54 , 7.56 ; 13C NMR  δ 27.3 (C-3), 28.2 (C2), 64.0 (C-1), 110.5 , 114.6 (C-9'), 118.5 (C-6), 118.6 (C-7), 121.5 , 127.1 (C-9), 127.3 (C-10), 128.2 , 135.3 (C-10'), 148.4 , 149.6 (C-8), 150.1 (C-5). ESI(+)-HRMS m/z 337.1076 [M + H]+ . For NMR spectra, see (+)-(R)-CJ-12,372 (15) (F26). max 226 (5.07), sh 207(4.94) nm. 1H NMR  δ 1.91 , 2.08 , 2.18 , 2.25 , 3.44 , 4.71 , 6.79 , 6.86 , 6.92 , 6.98 , 7.46 , 7.47 , 7.54 , 7.56 ; 13C NMR  δ 24.1 (C-2), 27.0 (C-3), 57.1 (OMe), 71.8 (C-1), 103.7 (C-4), 110.8 (C-7'), 110.8 (C-2'), 115.0 (C-9'), 118.9 (C-7), 119.2 (C-6), 121.6 (C-5'), 122.0 (C-4'), 122.0 (C-10), 125.7 (C-9), 128.5 (C-3'), 128.6 (C-6'), 135.7 (C-10'), 148.6 (C-1'), 149.1 (C-8'), 150.1 (C-8), 150.5 (C-5). ESI(+)-HRMS m/z 351.1227 [M + H]+ . For NMR spectra, (+)-(R)-Palmarumycin EG1 (16) (F29). max 226 (4.17), sh 214 (4.06) nm. 1H NMR  δ 2.02 , 2.12 , 2.14 , 2.20 , 3.47 , 4.73 , 6.85 , 6.92 , 6.93 , 7.26 , 7.27 , 7.43 , 7.45 , 7.50 , 7.51 ; 13C NMR  δ 23.8 (C-2), 26.1 (C-3), 56.8 (OMe), 71.6 (C-1), 101.1 (C-4), 109.9 , 114.5 (C-9'), 116.7 (C-6), 119.0 (C-8), 121.1 , 128.2 , 130.0 (C-5), 135.3 (C-10'), 148.9 (C-8'), 149.4 (C-1'). ESI(−)-HRMS m/z 333.1134 [M−H]− . For NMR spectra, see (R)-4-Methoxy-3,4-dihydro-2H-spirodioxin]-6-ol (17) (F32). max 224 (5.3), sh 26(4.4), 351 (4.2)nm. 1H NMR  δ 1.50 , 2.62 , 3.17 , 4.70 , 6.71 , 7.02 ; 13C NMR  δ 21.1 (C-11), 29.4 (C-4), 77.7 (C-3), 109.2 (C-9), 116.5 (C-7), 125.1 (C-6), 125.9 (C-10), 146.9 (C-5), 156.4 (C-8), 171.8 (C-1). ESI(+)-HRMS m/z 195.0652 [M + H]+ . For NMR spectra, see (R)-(-)-5-Hydroxymellein (18) (F19). R-MTPA ester derivative: to a stirred solution of 1  and dry deuterated pyridine-d5  in dry deuterochloroform CDCl3  at room temperature, (S)-(+)-MTPA-Cl  was added under inert atmosphere (Argon). The reaction was left for 2 h. The reaction mixture was then diluted in dry CDCl3 (0.6 ml) and submitted to 1H-NMR analysis. This gave the R-MTPA-methyl ester of 1 (34 mg) as a brown oil: 1H NMR  δ 1.31 , 3.44 , 3.94 , 4.17 , 4.54 , 5.12 , 5.91 , 6.03 , 6.11 , 6.71 ; 13C NMR  δ 13.7 (C-8), 54.3 (OMe), 64.0 (C6), 68.1 (C-5), 72.6 (C-7), 74.9 (C-2), 122.4 (C-4), 123.6 (C-2'), 130.5 (C-3), 138.7 (C-1').  and dry deuterated pyridine-d5  in dry deuterochloroform CDCl3 (100 μL [pyridine-d5] = 0.32 M) at room temperature, (R)-(-)-MTPA-Cl  was added under inert atmosphere (Argon). The reaction was left for 2 h. The reaction mixture was then diluted in dry CDCl3 (0.6 ml) and submitted to 1H-NMR analysis. This gave the S-MTPA-methyl ester of 1 (19.9 mg) as a brown oil: 1H NMR  δ 1.23 , 3.46 , 4.03 , 4.27 , 4.59 , 5.21 , 6.04 , 6.09 , 6.16 , 6.80 ; 13C NMR  δ 13.7 (C-8), 54.5 (OMe), 64.0 (C6), 68.2 (C-5), 72.4 (C-7), 75.3 (C-2), 122.7 (C-4), 123.8 (C-2'), 130.1 (C-3), 138.7 (C-1') ( (C-1'). . S-MTPA 7 (C-1') .https://doi.org/10.26037/yareta:k4p3ozyehbhg5nr4s7iuc5tkf4The raw NMR data files of the isolated compounds are available at the following link: 2 overnight. The next day, the medium in each well was replaced with 40 μl of the fresh one containing the indicated concentrations of compounds. After incubation for 3 days, the medium in each well was replaced with 25 μl of 0.5 mg/ml thiazolyl blue solution in 1xPBS followed by incubation for 3 h at 37°C. Then, the solution was removed and 25 μl DMSO was added to each well. Absorbance at 510 nm was measured in a Tecan Infinite M200 PRO plate reader.Indicated cell lines were detached and resuspended at 120,000 cells/ml and added to each well of a transparent 384-well plate in the final volume of 25 µl/well. The cells were maintained in DMEM containing 10% FBS at 37°C, 5% CO2 overnight for attachment. Subsequently, they were transfected by a plasmid encoding Renilla luciferase under the CMV promoter as described in the manufacturer’s protocol using 12 μg/ml of DNA and 40 μl/ml XtremeGENE 9 reagent. The next day, the medium in each well was replaced with a 20 μl fresh medium containing Wnt3a (2.5 μg/ml) or GSK3β inhibitor CHIR99021 (The Wnt-induced and basal luciferase activities were analyzed as described . BT-20 THIR99021 . After oHIR99021 .To measure the activity of luciferases, the culture medium was completely removed from all wells of the plate. Next, the luciferase activity of the firefly and Renilla luciferases was detected sequentially in individual wells of a 384-well plate through injection of corresponding measurement solutions in Infinite M Plex multifunctional plate reader with injection module .