MycoKeys 90: 85_| | 8 (2022) er-reviewed open-access journal doi: 10.3897/mycokeys.90.8287 | < MycoKkeys https://mycokeys.pensoft.net Launched to accelerate biodiversity research New polyketides from the liquid culture of Diaporthe breyniae sp. nov. (Diaporthales, Diaporthaceae) Blondelle Matio Kemkuignou!?, Lena Schweizer', Christopher Lambert'”, Elodie Giséle M. Anoumedem?, Simeon E Kouam?, Marc Stadler!*, Yasmina Marin-Felix!? | Department of Microbial Drugs, Helmholtz Centre for Infection Research (HZI) and German Centre for Infection Research (DZIF), Partner Site Hannover/Braunschweig, Inhoffenstrasse 7, 38124 Braunschweig, Germany 2. Institute of Microbiology, Technische Universitat Braunschweig, Spielmannstrafse 7, 38106 Braun- schweig, Germany 3 Department of Chemistry, Higher Teacher Training College, University of Yaoundé I, Yaoundé RO. Box 47, Cameroon Corresponding author: Yasmina Marin Felix (Yasmina.marinfelix@helmholtz-hzi.de) Academic editor: Thorsten Lumbsch | Received 9 March 2022 | Accepted 2 May 2022 | Published 14 June 2022 Citation: Matio Kemkuignou B, Schweizer L, Lambert C, Anoumedem EGM, Kouam SF, Stadler M, Marin-Felix Y (2022) New polyketides from the liquid culture of Diaporthe breyniae sp. nov. (Diaporthales, Diaporthaceae). MycoKeys 90: 85-118. https://doi.org/10.3897/mycokeys.90.8287 1 Abstract During the course of a study on the biodiversity of endophytes from Cameroon, a fungal strain was iso- lated. A multigene phylogenetic inference using five DNA loci revealed that this strain represents an un- described species of Diaporthe, which is introduced here as D. breyniae. Investigation into the chemistry of this fungus led to the isolation of two previously undescribed secondary metabolites for which the trivial names fusaristatins G (7) and H (8) are proposed, together with eleven known compounds. ‘The struc- tures of all of the metabolites were established by using one-dimensional (1D) and two-dimensional (2D) Nuclear Magnetic Resonance (NMR) spectroscopic data in combination with High-Resolution Electro- Spray Ionization Mass Spectrometry (HR-ESIMS) data. The absolute configuration of phomopchalasin N (4), which was reported for the first time concurrently to the present publication, was determined by analysis of its Rotating frame Overhauser Effect SpectroscopY (ROESY) spectrum and by comparison of its Electronic Circular Dichroism (ECD) spectrum with that of related compounds. A selection of the isolated secondary metabolites were tested for antimicrobial and cytotoxic activities, and compounds 4 and 7 showed weak antifungal and antibacterial activity. On the other hand, compound 4 showed moder- ate cytotoxic activity against all tested cancer cell lines with IC,, values in the range of 5.8-45.9 uM. The latter was found to be less toxic than the other isolated cytochalasins (1-3) and gave hints in regards to the structure-activity relationship (SAR) of the studied cytochalasins. Fusaristatin H (8) also exhibited weak cytotoxicity against KB3.1 cell lines with an IC,, value of 30.3 uM. Copyright Blondelle Matio Kemkuignou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 86 Blondelle Matio Kemkuignou et al. / MycoKeys 90: 85-118 (2022) Graphical abstract T fe) “oy fe) fe) fe) eh Diaporthe breyniae tie “+. sp. nov. iil 8 Keywords Antimicrobial, cytotoxicity, Diaporthe, endophytic fungi, one new species, secondary metabolites Introduction The genus Diaporthe (including their asexual states, which were previously referred to as Phomopsis spp.) comprises several hundred species mostly attributed to plant pathogens, non-pathogenic endophytes, or saprobes in terrestrial host plants (Chep- kirui and Stadler 2017; Xu et al. 2021). The term “endophytic fungi” herein refers to a group of microorganisms that inhabit the internal parts of a plant, but typically cause no apparent symptoms of disease in the host plant (Stone et al. 2000). Fungal endophytes belonging to the genus Diaporthe have been widely investigated by natural product chemists and have proven to be a rich source of novel organic compounds with interesting biological activities and a high level of chemical diversity (Chepkirui and Stadler 2017). They have been shown to predominantly produce polyketides, but PKS/NRPS-derived hybrids like cytochalasins have also been frequently reported from Diaporthe (Jouda et al. 2016; Chepkirui and Stadler 2017). Initially, cytochalasins have been discovered for their potent cytotoxic effects, which are due to their interference with the actin cytoskeleton (Yahara et al. 1982) and have been targeted primarily as anticancer agents. However, not all cytochalasins are equally active on actin (Kretz et al. 2019), and they were even found to significantly inhibit biofilm formation of an important human pathogenic bacterium (Yuyama et al. 2018). The current paper supports the activities of an interdisciplinary consortium that aims at exploring the New polyketides from Diaporthe breyniae 87 chemical space of the cytochalasins, in order to establish structure-activity relation- ships (SAR) and systematically explore their utility for application in various medical applications. Owing to the structural complexity of cytochalasins, their total synthesis remains tedious and requires several reaction steps with relatively low final yields (Zag- houani et al. 2016; Long et al. 2018). Moreover, most of the compounds that were re- ported previously have not been studied thoroughly for their biological effects; hence, it is worth obtaining them from the fungal producer organisms by de novo isolation and characterization. We have recently isolated and studied a new endophytic species of Diaporthe from the twigs of Breynia oblongifolia. We noted prominent antimicrobial effects in the ex- tracts derived from this strain and decided to study its secondary metabolites. The cur- rent paper includes the description of the new species D. breyniae sp. nov., and reports details on the isolation and structure elucidation of its secondary metabolites, as well as an account of their biological properties. Materials and methods Fungal isolation The fungus was isolated from fresh twigs of an apparently healthy plant belonging to Breynia oblongifolia in Kala Mountain (Yaoundé, Cameroon). Fresh twigs (5 x 5 cm length) of Breynia oblongifolia were thoroughly washed with running tap water, then disinfected in 75% ethanol for 1 min, in 3% sodium hypochlorite (NaClO) for 10 min, and finally in 75% ethanol for 30 s. These twigs were then rinsed three times in sterile distilled water and dried on sterile tissue paper under a laminar flow hood. Small segments of the twigs were transferred to Petri dishes containing potato dextrose agar (PDA, HiMedia, Mumbai, India) supplemented with 100 mg/mL penicillin and 100 pg/mL streptomycin sulphate and incubated at 28 °C. After 10 days, fungal colo- nies were examined and hyphal tips were transferred to PDA using a sterile needle and incubated at 28 °C. Herbarium type material and the ex-type strain of the new species are maintained at the collection of the Westerdijk Fungal Biodiversity Institute (CBS), Utrecht, the Netherlands. Phenotypic study For cultural characterization, the isolate was grown for 15 days on malt extract agar (MEA; HiMedia, Mumbai, India), oatmeal agar (OA; Sigma-Aldrich, St. Louis, Mis- souri, USA), and PDA at 21 °C in darkness (Guarnaccia et al. 2018). Color notations in parentheses are taken from the color chart of The Royal Horticultural Society Lon- don (1966). The fungus was grown in 2% tap water agar supplemented with sterile pine needles (PNA; Smith et al. 1996) to induce sporulation. 88 Blondelle Matio Kemkuignou et al. / MycoKeys 90: 85-118 (2022) Molecular study DNA of the fungus was extracted and purified directly from colony growing in yeast malt agar (YM agar; malt extract 10 g/L, yeast extract 4 g/L, D-glucose 4 g/L, agar 20 g/L, pH 6.3 before autoclaving), following the Fungal g DNA Miniprep Kit EZ-10 Spin Column protocol (NBS Biologicals, Cambridgeshire, UK). The amplification of the ITS, cal, his3, tefl and tub2 loci were performed according to White et al. (1990) (ITS), Carbone and Kohn (1999) (caland tef1), Glass and Donaldson (1995) (Ais3 and tub2) and Crous et al. (2004) (/7s3). PCR products were purified and sequenced us- ing Sanger Cycle Sequencing method at Microsynth Seqlab GmbH (Gottingen, Ger- many), and the consensus sequences obtained employing the de-novo assembly feature of the Geneious 7.1.9 (http://www.geneious.com, Kearse et al. 2012) program package using a forward and reverse read. In order to restrict the phylogenetic inference to the relevant species to com- pare with, a first phylogenetic analysis was carried out based on the combination of the five loci sequences (ITS, cal, his3, tefl, tub2) of our isolate and a selection of sequence data derived from type material or reference strains from all Diaporthe spp. available in NCBI. Each locus was aligned separately using MAFFT v. 7.017 (algorithm G-INS-I, gap open penalty set to 1.53, offset value 0.123 with options set for automatically determining sequence direction automatically and more ac- curately) as available as a Geneious 7.1.9 plugin (Katoh and Standley 2013) and manually adjusted in MEGA v. 10.2.4 (Kumar et al. 2018). Alignment errors were minimized by using gblocks (Talavera and Castresana 2007); with options set for allowed block positions ‘with half’, minimum length of a block set to 5 and a maxi- mum of 10 contiguous nonconserved positions) and concatenated by employing the phylosuite v 1.2.2 program package (Zhang et al. 2020). Maximum-Likelihood tree inference followed using IQTree V2.1.3 (Minh et al. 2020) preceded by calcu- lation and automatic selection of the appropriate nucleotide exchange model us- ing ModelFinder (Chernomor et al. 2016; Kalyaanamoorthy et al. 2017) based on Bayesian inference criterion. Bootstrap support was calculated by parallelizing 10 independent maximum-likelihood (ML) tree searches with 100 bootstrap replicates each to minimize computational burden. The total 1000 bootstrap replicates were consequently mapped onto the ML tree with the best (highest) ML score. After selection of the core group related to the sequences derived from D. breyniae sp. nov., a second phylogenetic analysis was performed including all five sequenced loci, using D. amygdali CBS 126679! and D. eres CBS 138594! as outgroups. Sequence alignment and curation steps were identical, with exemption of a manual curation instead of employing automatic filtering for misaligned alignment sections using gblocks. ML trees using the supermatrix and single loci, respectively, were inferred using IQ Tree 2.1.3 with ModelFinder to determine optimal substitution models for each loci and partition, using 1000 bootstrap replicates to assign statistical support. The clade in which the sequences of the novel strain clustered, was checked visu- ally for congruence among the single locus trees. Concurrently, a second tree was New polyketides from Diaporthe breyniae 89 inferred following a Bayesian approach using MrBayes 3.2.7a (Ronquist et al. 2012) with nucleotide substitution models previously determined using PartitionFinder2 (Lanfear et al. 2016, options set for unlinked partitions, BIC, restricting models for Bayesian inference) and concatenated in Phylosuite V.1.2.2. Bayesian inference was done in Mr. Bayes v. 3.2.7 (Ronquist et al. 2012), using Markov Chain Monte Carlo (MCMC) with four incrementally heated chains (temperature parameter set to 0.15), starting from a random tree topology. Generations were set to 100.000.000 with convergence controlled by average standard deviation of split frequencies ar- riving below 0.01. Trees were sampled every 1000 generations with the first 25% of saved trees treated as “burn-in” phase. Posterior probabilities were mapped using the remaining trees. Bootstrap support (bs) = 70 and posterior probability values (pp) > 0.95 were considered significant (Alfaro et al. 2003). The sequences generated in this study are deposited in GenBank (Table 1) and the alignments used in the phy- logenetic analysis are included in Supplementary material. Sequences retrieved from GenBank are indicated in Table 1 and Suppl. material 1: $4. Chromatography and spectral methods Electrospray ionization mass (ESIMS) spectra were recorded with an UltiMate 3000 Series uHPLC (Thermo Fischer Scientific, Waltman, MA, USA) utilizing a C18 Acquity UPLC BEH column (2.1 x 50 mm, 1.7 um; Waters, Milford, USA) con- nected to an amaZon speed ESI-Iontrap-MS (Bruker, Billerica, MA, USA). HPLC parameters were set as follows: solvent A: H,O + 0.1% formic acid, solvent B: acetoni- trile (ACN) + 0.1% formic acid, gradient: 5% B for 0.5 min increasing to 100% B in 19.5 min, then isocratic condition at 100% B for 5 min, a flow rate of 0.6 mL/min, and Diode-Array Detection (DAD) of 210 nm and 190-600 nm. High-resolution electrospray ionization mass spectrometry (HR-ESIMS) spectra were recorded with an Agilent 1200 Infinity Series HPLC-UV system (Agilent Tech- nologies, Santa Clara, USA; column 2.1 x 50 mm, 1.7 um, C18 Acquity UPLC BEH (waters), solvent A: H,O +0.1% formic acid; solvent B: ACN + 0.1% formic acid, gradient: 5% B for 0.5 min increasing to 100% B in 19.5 min and then maintaining 100% B for 5 min, flow rate 0.6 mL/min, UV/Vis detection 200—640 nm) connected to a MaXis ESI-TOF mass spectrometer (Bruker) (scan range 100-2500 m/z, capillary voltage 4500 V, dry temperature 200 °C). Optical rotations were recorded in methanol (Uvasol, Merck, Darmstadt, Ger- many) by using an Anton Paar MCP-150 polarimeter (Seelze, Germany) at 20 °C. UV/Vis spectra were recorded using methanol (Uvasol, Merck, Darmstadt, Germany) with a Shimadzu UV/Vis 2450 spectrophotometer (Kyoto, Japan). ECD spectra were obtained on a J-815 spectropolarimeter (JASCO, Pfungstadt, Germany). Nuclear magnetic resonance (NMR) spectra were recorded at a temperature of 298 K with an Avance III 500 spectrometer (Bruker, Billerica, MA/USA, 'H-NMR: 500 MHz and "C-NMR: 125 MHz) and an Ascend 700 spectrometer with 5 mm TCI cryoprobe (Bruker, Billerica, MA/USA, 'H-NMR: 700 MHz and °C-NMR: 175 MHz). 90 Blondelle Matio Kemkuignou et al. / MycoKeys 90: 85-118 (2022) Table |. Isolated and reference strains of Diaporthe included in this study. # GenBank accession numbers in bold were newly generated in this study. The taxonomic novelty is indicated in bold italic. Species Diaporthe acaciarum D. acericola dS alangii ambigua amygdali angelicae arctii Se&Soyy arezzoensis batatas beilharziae D. biguttulata D. breyniae D. camporesii D. caryae D. celtidis D. cerradensis D. chimonanthi D. chinensis D. chromolaenae D. cichorii D. cinnamomi citriasiana compacta convolvuli SS&ss cucurbitae dS cuppatea D. discoidispora D. durionigena D. endophytica D. eres D. fici-septicae D. fructicola D. ganjae D. glabrae D. goulteri Isolates! CBS 138862" MFLUCC 17-0956" CFCC 52556! CBS 1140157 CBS 126679" CBS 1115927 CBS 136.25 MELU 19- 2880" CBS 122.21 BRIP 54792" ICMP 20657" CBS 1489107 JZB 320143" CFCC 52563! NCYU 19- 0357" CMRP 43317 SCHM 3614" MFLUCC 19-0101" MFLUCC 17-1422" MFLUCC 17-1023" CFCC 52569! CBS 1342407 LC3083" CBS 124654 DAOM 42078" CBS 1174997 ICMP 206627 VTCC 930005° CBS 1338117 CBS 138594? MELU 18- 2588" MAFF 246408! CBS 180.917 SCHM 36227 BRIP 55657a" ITS KP004460 KY964224 MH121491 KC343010 KC343022 KC343026 KC343031 MT185503 KC343040 JX862529 KJ490582 ON400846 MN533805 MH121498 MW114346 MN173198 AY622993 MW187324 MH094275 KY964220 MH121504 JQ954645 KP267854 KC343054 KM453210 AY339322 kJ490624 MN453530 KC343065 kJ210529 MW114348 LC342734 KC343112 AY601918 kJ197290 GenBank accession numbers? tub2 KP004509 KY964074 MH121573 KC343978 KC343990 KC343994 KC343999 MT454055 KC344008 KF170921 KJ490403 ON409186 MNS561316 MH121580 MW148266 MW751671 MW245013 KY964104 MH121586 KC357459 KP293434 KC344022 KP118848 JX275420 KJ490445 MT276159 KC344033 kJ420799 MW148268 LC342736 KC344080 KJ197270 his3 KP004504 MH121451 KC343494 KC343506 KC343511 KC343515 KC343524 KJ490524 ON409187 MH121458 MW751663 MH121464 MF418282 KP293508 KC343538 KM453212 KC343541 KJ490566 KC343549 KJ420850 LC342737 KC343596 refi KY964180 MH121533 KC343736 KC343748 KC343752 KC343757 KC343766 JX862535 KJ490461 ON409188 MH i 1540 MW192209 MT311685 MW205017 KY964176 MH121546 JQ954663 KP267928 KC343780 KM453211 AY339354 KJ490503 MT276157 KC343791 kJ210550 MW192211 LC342735 KC343838 KJ197252 KY964137 MH121415 KC343252 KC343264 KC343268 KC343273 KC343282 ON409189 MH121422 MW751655 MW294199 KY964133 KC357491 KC343296 JX197414 KC343307 KJ434999 LC342738 KC343354 References Crous et al. (2014) Dissanayake et al. (2017) Yang et al. (2018) Gomes et al. (2013) Gomes et al. (2013) Gomes et al. (2013) Gomes et al. (2013) Li et al. (2020) Gomes et al. (2013) Thompson et al. (2015) Huang et al. (2015) Present study Hyde et al. (2020) Yang et al. (2018) Tennakoon et al. (2021) Iantas et al. (2021) Chang et al. (2005) de Silva et al. (2021) Mapook et al. (2020) Dissanayake et al. (2017) Yang et al. (2018) Huang et al. (2013) Gao et al. (2016) Gomes et al. (2013) Udayanga et al. (2015) Van Rensburg et al. (2006) Huang et al. (2015) Crous et al. (2020) Gomes et al. (2013) Udayanga et al. (2014) Tennakoon et al. (2021) Crous et al. (2019) Gomes et al. (2013) Chang et al. (2005) Thompson et al. (2015) Species D. guangdongensis D. gulyae D. guttulata D. helianthi D. heterostemmatis D. hordei D. hubeiensis D. infecunda D. infertilis D. kochmanii D. kongii leucospermi longicolla longispora lusitanicae SSsyus machili S manihotia S masirevicit . maytent . megalospora D D D. melonis D. micheliae D . middletonii D. myracrodruonis D. neoarctii neoraonikayaporum D. novem D. ovalispora D. pachirae D. passifloricola D. phaseolorum D. pseudolongicolla D. pyracanthae D. racemosae D. raonikayaporum D. rosae rosiphthora rossmaniae SoD sackstonii D. sambucusii Isolates! ZHKUCC20- 0014" BRIP 54025 CGMCC 3.201007 CBS 592.817 SAUCC 194.857 CBS 481.92 JZB 320123" CBS 133812? CBS 230.527 BRIP 54033? BRIP 540317 CBS 1119807 FAU 599™ CBS 194.36" CBS 1232127 SAUCC 194.1117 CBS 505.76 BRIP 57892a! CBS 1331857 CBS 143.27 CBS 507.78" SCHM 3603 BRIP 54884e! URM 7972! CBS 109490 MFLUCC 14-1136" CBS 127271" ICMP 20659" COAD 2074" CBS 141329° CBS 113425 CBS 117165" CBS142384! CBS 143770° CBS 133182" MFLUCC 17-2658" COAD 2913" CAA 762° BRIP 54669b" CFCC 51986" New polyketides from Diaporthe breyniae ITS MT355684 JF431299 MT385950 KC343115 MT822613 KC343120 MK335809 KC343126 KC343052 JF431295 JF431301 JN712460 kJ590728 KC343135 KC343136 MT822639 KC343138 KJ197277 KC343139 KC343140 KC343142 AY620820 KJ197286 MK205289 KC343145 KU712449 KC343157 kJ490628 MG559537 KX228292 KC343174 DQ286285 KY435635 MG600223 KC343188 MG828894 MT311196 MK792290 KJ197287 KY852495 GenBank accession numbers? tub2 MT409292 KJ197271 MT424705 KC344083 MT855810 KC344088 MK500148 KC344094 KC344020 KJ197272 KY435673 KJ610883 KC344103 KC344104 MT855836 KC344106 KJ197257 KC344107 KC344108 KC344110 KJ197266 MK205291 KC344113 KU743988 KC344125 KJ490449 MG559541 KX228387 KC344142 KY435666 MG600227 KC344156 MG843878 MK837914 KJ197267 KY852511 his3 MW022491 KC343599 MT855581 KC343604 KC343610 KC343536 KY435653 KJ659188 KC343619 KC343620 MT855606 KC343622 KC343623 KC343624 KC343626 KC343629 KC343641 KJ490570 KX228367 KC343658 KY435645 MGG600221 KC343672 MK871432 KY852503 ‘efi MT409338 JN645803 MT424685 KC343841 MT855925 KC343846 MK523570 KC343852 KC343778 JN645809 JN645797 KY435632 KJ590767 KC343861 KC343862 MT855951 KC343864 KJ197239 KC343865 KC343866 KC343868 KJ197248 MK213408 KC343871 KU749369 KC343883 KJ490507 MG559539 KC343900 DQ286259 KY435625 MG600225 KC343914 MT313692 MK828063 kJ197249 KY852507 cal MT409314 MW022470 JX197454 MT855692 KC343362 MK500235 KC343368 KC343294 KY435663 KJ612124 KC343377 KC343378 MT855718 KC343380 KC343381 KC343382 KC343384 MK205290 KC343387 KU749356 KC343399 MG559535 KC343416 KY435656 MG600219 KC343430 MG829273 MT313690 MK883822 KY852499 91 References Dong et al. (2021) Thompson et al. (2015) Dissanayake et al. (2020) Gomes et al. (2013) Sun et al. (2021) Gomes et al. (2013) Manawasinghe et al. 2019 Gomes et al. (2013) Guarnaccia and Crous (2017) Thompson et al. (2011) Thompson et al. (2011) Crous et al. (2011c) Udayanga et al. (2015) Gomes et al. (2013) Gomes et al. (2013) Huang et al. (2021) Gomes et al. (2013) Thompson et al. (2015) Gomes et al. (2013) Gomes et al. (2013) Gomes et al. (2013) Chang et al. (2005) Thompson et al. (2015) da Silva et al. (2019) Gomes et al. (2013) Doilom et al. (2017) Gomes et al. (2013) Huang et al. (2015) Milagres et al. (2018) Crous et al. (2016) Gomes et al. (2013) Petrovié et al. (2018) Santos et al. (2017) Marin-Felix et al. (2019) Gomes et al. (2013) Wanasinghe et al. (2018) Pereira et al. (2021) Hilario et al. (2020) Thompson et al. (2015) Yang et al. (2018) 92 D. D. Species schini schoeni sclerotioides serafiniae Siamensis , sinensis sojae stewartit subellipicola subordinaria tecomae tectonae tectonendophytica D. D. D. D. D. Dz. Dz. D. terebinthifolii thunbergiicola tulliensis ueckeri unshiuensis vexans vitimegaspora vochysiae D. yunnanensis Blondelle Matio Kemkuignou et al. / MycoKeys 90: 85-118 (2022) Isolates! CBS 133181" MELU 15- 1279? CBS 296.67" BRIP 55665a! MFLUCC 10-0573a CGMCC 3.195217 CBS 139282" CBS 193.36 KUMCC 17201532 CBS 101711 CBS 100547 MFLUCC 12-0777" MFLUCC 13-04717 CBS 133180! MFLUCC 12-0033" BRIP 62248a FAU 656 BRIP 54736j (type of D. miriciae) CGMCC 3.17569! CBS 127.14 STE-U 2675 LGMEF 1583! CGMCC 3.182897 ITS KC343191 KY964226 KC343193 KJ197274 JQ619879 MK637451 KJ590719 FJ889448 MG746632 KC343213 KC343215 KU712430 KU712439 KC343216 KP715097 KR936130 KJ590726 kJ197283 KJ490587 KC343229 AF230749 MG976391 KX986796 GenBank accession numbers? tub2 KC344159 KY964109 KC344161 KJ197254 JX275429 MK660447 K]610875 MG746634 KC344181 KC344183 KU743977 KU743986 KC344184 KR936132 KJ610881 KJ197263 KJ490408 KC344197 MK007527 KX999228 his3 KC343675 KC343677 KJ659208 KC343697 KC343699 KC343700 KJ659215 KJ490529 KC343713 MK033323 KX999267 ‘efi KC343917 KY964182 KC343919 KJ197236 JX275393 MK660449 KJ590762 GQ250324 MG746633 KC343939 KC343941 KU749359 KU749367 KC343942 KP715098 KR936133 KJ590747 KJ197245 KJ490466 KC343955 MK007526 KX999188 cal KC343433 KY964139 KC343435 KJ612116 KC343455 KC343457 KU749345 KU749354 KC343458 KJ612122 KC343471 MK007528 KX999290 References Gomes et al. (2013) Dissanayake et al. (2017a) Gomes et al. (2013) Thompson et al. (2015) Udayanga et al. (2012) Feng et al. (2019) Udayanga et al. (2015) Santos et al. (2010) Hyde et al. (2018) Gomes et al. (2013) Gomes et al.(2013) Doilom et al. (2017) Doilom et al. (2017) Gomes et al. (2013) Liu et al. (2015) Crous et al. (2015) Huang et al. (2015) Thompson et al. (2015) Huang et al. (2015) Gomes et al.(2013) Mostert et al. (2001) Noriler et al. (2019) Gao et al. (2017) 'BRIP: Queensland Plant Pathology Herbarium, Brisbane, Australia; CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands; CGMCC: Chinese General Microbiological Culture Collection Cen- ter, Beijing, China; COAD: Culture Collection of Octavio de Almeida Drumond. Universidade Federal de Vicosa, Vicosa, Brasil; FAU: Isolates in culture collection of Systematic Mycology and Microbiology Laboratory; ICMP: International Collection of Micro-organisms from Plants, Auckland, New Zealand; KUMCC: Kumming Institute of Botany, Kumming, China; LGME, Laboratorio de Genética de Micro- rganismos (LabGeM) culture collection, at the Federal University of Parana, Brazil; MAFF: Ministry of Agriculture, Forestry and Fisheries, Tokyo, Japan; MFLUCC: Mae Fah Luang University Culture Collec- tion, Chiang Rai, Thailand; SAUCC: Shandong Agricultural University Culture Collection, Shandong, China; STE-U: Department of Plant Pathology, Stellenbosch University, Stellenbosch, South Africa; URM: Culture Collection at the Universidade Federal de Pernambuco, Recife, Brazil; VIT'CC: Vietnam Type Culture Collection, Center of Biotechnology, Vietnam National University, Hanoi, Vietnam; ZH- KUCC: Culture Collection of Zhongkai University of Agriculture and Engineering, Guangzhou, China. T indicates type material. “ITS: internal transcribed spacers and intervening 5.8S nrDNA; tub2: partial 6-tubulin gene; /is3: partial histone H3 gene; tef/: partial elongation factor 1-alpha gene; ca/: partial calmodulin gene. New polyketides from Diaporthe breyniae 93 Small-scale fermentation and extraction The fungus was cultivated in three different liquid media (YM 6.3 medium: 10g/mL malt extract, 4¢/mL, yeast extract, 4g/mL, D-glucose and pH = 6.3, Q6 % medium: 10 g/mL glycerin, 2.5 g/mL D-glucose, 5 g/mL cotton seed flour and pH = 7.2; ZM ¥2 medium: 5 g/mL molasses, 5 g/mL oatmeal, 1.5 g/mL D-glucose, 4 g/mL sac- charose, 4 g/mL mannitol, 0.5 g/mL edamin, ammonium sulphate 0.5 g/mL, 1.5 g/ mL calcium carbonate and pH = 7.2) (Chepkirui et al. 2016). A well-grown 14-day- old mycelial culture grown on YM agar was cut into small pieces using a cork borer (7mm), and five pieces used for inoculation of 500 mL Erlenmeyer flasks containing 200 mL of media. The cultures were incubated at 23 °C on a rotary shaker at 140 rpm. The growth of the fungus was monitored by checking the amount of free glucose daily using Medi-Test glucose strips (Macherey Nagel, Diiren, Germany). The fermentation was terminated three days after glucose depletion and the biomasses and supernatants were separated via vacuum filtration. Afterwards, the supernatants were extracted with equal amount of ethyl acetate (200 mL) and filtered through anhydrous sodium sul- phate. The resulting ethyl acetate extracts were evaporated to dryness in vacuo (Rotary Evaporator: Heidolph Instruments GmbH & Co. KG, Schwabach, Germany; pump: Vacuubrand GmbH & Co. KG, Wertheim am Main, Germany) at 40 °C. The mycelia were extracted with 200 mL of acetone in an ultrasonic bath (Sonorex Digital 10 P, Bandelin Electronic GmH & Co. KG, Berlin, Germany) at 40 °C for 30 min, filtered and the organic phase evaporated. The volume of the remaining aqueous phase was adjusted with an equal amount of distilled water and subjected to the same procedure as described for the supernatants. The small-scale cultivation of Diaporthe breyniae was also carried out on YM agar medium and rice solid medium (BRFT, brown rice 28 g as well as 0.1 L of base liquid (yeast extract 1 g/L, di-sodium tartrate di-hydrate 0.5 g/L, KH,PO, 0.5 g/L) (Becker et al. 2020a). Briefly, the fungus was grown on a YM agar plate and the mycelia was extracted with 200 mL of ethyl acetate in an ultrasonic water bath at 40 °C for 30 min, filtered and the filtrate evaporated to dryness in vacuo at 40 °C. For BFRT medium, three small pieces of the mycelial culture grown on a YM agar plate were inoculated into a 250 ml Erlenmeyer flask containing 100 mL of YM 6.3 medium. The seed cul- ture was incubated at 23 °C under shake condition at 140 rpm. After 5 days, 10 mL of this seed culture were transferred to a 500 mL Erlenmeyer flask containing BRFT medium and incubated for 28 days at 23 °C. Afterwards, extraction of the culture was performed following the same procedure as above mentioned for the mycelia obtained from the liquid cultures. Scale-up fermentation in shake flask batches and extraction Preliminary results obtained from small-scale screening suggested that the fungus grew and produced best in ZM ¥2 medium (Suppl. material 1: Figs $1, $2). Moreover, the extracts obtained from the fungal culture in ZM % were active against Bacillus subtilis 94 Blondelle Matio Kemkuignou et al. / MycoKeys 90: 85-118 (2022) and Mucor plumbeus. Therefore, this medium was selected for scale-up fermentation. Three well-grown 14-day-old YM agar plate of the mycelial culture were cut into small pieces using a 7 mm cork borer and 5 pieces inoculated in 10 x 500 mL Erlenmeyer flasks containing 200 mL of ZM % medium. The culture was incubated at 23 °C on a rotary shaker at 140 rpm for 11 days. Fermentation was aborted 3 days after the depletion of free glucose. The mycelia and supernatant from the batch fermentation were separated via vacuum filtration. The mycelia were extracted with 3 x 500 mL of acetone in an ultrasonic water bath at 40 °C for 30 min. The extracts were combined and the solvent evaporated in vacuo (40 °C). The remaining water phase was subjected to the same procedure as previously described for the mycelial fraction in small-scale extraction, repeating the extraction step 3 times, yielding 955 mg dark brown solid- like extract. The supernatant (2 L) was extracted with equal amount of ethyl acetate and filtered through anhydrous sodium sulphate. ‘The resulting ethyl acetate extract was evaporated to dryness in vacuo to afford 251 mg of extract. Isolation of secondary metabolites The mycelial and the supernatant extracts from shake flask batch fermentation dis- solved in methanol were centrifuged by means of a centrifuge (Hettich Rotofix 32 A, Tuttlingen, Germany) for 10 min at 4000 rpm. Afterwards, the mycelia and supernatant extracts were fractionated separately using preparative reverse phase HPLC (Biichi, Pure C-850, 2020, Switzerland). VP Nucleodur 100-5 C18ec col- umn (150 x 40 mm, 7 um: Machery-Nagel, Diiren, Germany) was used as sta- tionary phase. Deionized water (Milli-Q, Millipore, Schwalbach, Germany) sup- plemented with 0.1% formic acid (FA) (solvent A) and acetonitrile (ACN) with 0.1% FA (solvent B) were used as the mobile phase. The elution gradient used for fractionation was 5—35% solvent B for 20 min, 35-80% B for 30 min, 80—100% B for 10 min and thereafter isocratic condition at 100% solvent B for 15 min. The flow rate was set to 30 mL/min and UV detection was carried out at 210, 320 and 350 nm. For the supernatant extract, 13 fractions (F1-F13) were selected according to the observed peaks, and further analysis of the fractions using HPLC-MS revealed that four of the obtained fractions constituted pure compounds. Using the same elution conditions as mentioned, the mycelia extract afforded 17 fractions (F1—F17) selected from the observed peaks. HPLC-MS analysis of the obtained fractions re- vealed that seven fractions constituted pure compounds. The compounds obtained from mycelial and supernatant extracts were combined according to their respec- tive HPLC-ESIMS retention time and molecular weight. Compound 1 (55.2 mg, t, = 7.80 min) was obtained from both the mycelium and supernatant extracts as well as compounds 2 (10.9 mg, ¢, = 6.27 min), 3 (2.6 mg, ¢, = 11.42 min) and 4 (5.6 mg, ¢, = 9.49 min). Compounds 5 (3.6 mg, ¢, = 13.46 min), 11 (0.7 mg, ¢, = 12.11 min) and 12 (2.0 mg, ¢, = 3.83 min) were only isolated from the myce- lial extract. Fractions F4 from both the mycelium and supernatant extracts were combined and purified using an Agilent Technologies 1200 Infinity Series semi- New polyketides from Diaporthe breyniae 95 preparative HPLC instrument (Waldbronn, Germany). The elution gradient used was 20-30% solvent B for 5 min followed by isocratic condition at 30% B for 25 min and thereafter increased gradient from 30—-100% B for 5 min. VP Nucleodur 100-5 C18ec column (250 x 10 mm, 5 ym: Machery-Nagel, Diiren, Germany) was used as stationary phase and the flow rate was 3 mL/min. These fractions afforded compound 13 (2.34 mg, t,= 5.13 min). Fractions F13 and F14 from the mycelial extract were combined with F12 from the supernatant as they contained the same compounds. The pooled fractions were purified by preparative reverse phase HPLC (Biichi, Pure C-850, 2020, Switzerland). VP Nucleodur 100-5 C18ec column (250 x 21 mm, 5 um: Machery-Nagel, Diiren, Germany) was used as stationary phase with a flow rate of 15 mL/min and an elution gradient of 5—70% solvent B for 5 min, followed by isocratic conditions at 70% B for 25min, and thereafter increased gradient from 70—100% B for 5 min. These fractions afforded compound 9 (10.5 mg, ¢, = 13.02 min) and sub-fraction G1. Sub-fraction G1 was further purified us- ing an Agilent Technologies 1200 Infinity Series semi-preparative HPLC with the elution gradient starting from 65-70% B for 5 min followed by isocratic condition at 70% B for 25 min and thereafter increased gradient from 70—100% B for 5 min to afford compounds 7 (1.4 mg, ¢,= 13.91 min) and 8 (0.52 mg, ¢, = 13.56 min). Fraction F15 from the mycelium were also purified using the same instrument and same elution conditions as described for sub-fraction G1. This fraction afforded compounds 6 (1.1 mg, ¢, = 14.02 min) and 10 (1.7 mg, ¢, = 13.58 min). Note: The given retention times were obtained from HPLC-ESIMS following the HPLC parameters as described in the general experimental procedures. Antimicrobial assay The antifungal and antibacterial activities (Minimum Inhibition Concentration, MIC) of all extracts obtained from small-scale fermentation were determined in serial dilution assays as described previously (Chepkirui et al. 2016; Becker et al. 2020b) against Bacillus subtilis, Candida tenuis, Escherichia coli and Mucor plumbeus. The assays were carried out in 96-well microtiter plates in YM 6.3 medium for fila- mentous fungi and yeast and MHB medium (Miiller-Hinton Broth: SN X927.1, Carl Roth GmbH, Karlsruhe, Germany) for bacteria. Starting concentration for all extracts were 300 ug/mL. In addition, the antimicrobial activity of the isolated pure compounds was also assessed as previously described (Matio Kemkuignou et al. 2020) against a panel of bacteria and fungi including Pichia anomala DSM 6766, Schizosaccharomyces pombe DSM 70572, Mucor hiemalis DSM 2656, Candida albicans DSM 1665, and Rhodotorula glutinis DSM 10134 for fungal microorgan- isms, Bacillus subtilis DSM 10, Staphyloccocus aureus DSM 346 and Mycobacterium smegmatis ATCC 700084 for Gram-positive bacteria, Acinetobacter baumannii DSM 30008, Chromobacterium violaceum DSM 30191, Escherichia coli DSM 1116 and Pseudomonas aeruginosa for Gram-negative bacteria. Starting concentration for tested compounds was adjusted to 66.7 g/mL. 96 Blondelle Matio Kemkuignou et al. / MycoKeys 90: 85-118 (2022) Cytotoxicity assay The in vitro cytotoxicity (IC,,) of the isolated metabolites against several mammalian cell lines (human endocervical adenocarcinoma KB 3.1, mouse fibroblasts L929, squa- mous cancer A431, breast cancer MCF-7, lung cancer A549, ovary cancer SK-OV-3 and prostate cancer PC-3) was determined by colorimetric tetrazolium dye MTT as- say using epothilone B as a positive control in accordance to our previously reported experimental procedure (Becker et al. 2020b). Results and discussion Phylogenetic study The lengths of the fragments of the first phylogenetic inference using the five previously mentioned loci used in the combined dataset for the tree including all Diaporthe spp. were 454 bp (ITS), 318 bp (cal), 296 bp (4is3), 153 bp (zefZ) and 487 bp (tub2), comprising in total 341 taxa. The length of the final alignment was 1708 bp. ‘The inferred phylogeny with the best maximum likelihood score with bootstrap support (bs) values mapped onto branch bipartitions is shown in Suppl. material 1: Fig. S100. The here studied strain was located in a clade with 92% bs including 341 taxa, including species belonging to the D. sojae complex. A second molecular phylogeny was inferred including sequences of the same loci, but restricted to the aforementioned clade, including 98 taxa. ‘The lengths of the fragments used in the combined dataset were 572 bp (ITS), 449 bp (cal), 373 bp (his3), 452 bp (tef1) and 862 bp (tub2), totaling 2708 bp for the final alignment. Fig. 1 shows the consensus ML tree, including bs and Bayesian posterior probability (pp) values at the nodes. Our strain was located in an independent branch distant from other spe- cies of Diaporthe, demonstrating that this represented a new species, which is introduced here as D. breyniae. Unfortunately, the new species lacked sporulation in all media tested in the present study. Therefore, the introduction of it is based only on molecular data. Taxonomy Diaporthe breyniae Y. Marin & C. Lamb., sp. nov. MycoBank No: 843243 Etymology. Name refers to the host genus that this fungus was isolated from, Breynia. Description. Not sporulated. Diaporthe breyniae differs from its closest phyloge- netic neighbour, D. durionigena by unique fixed alleles in three loci based on alignments of the separate loci included in the supplementary material: ITS positions 93 (indel), 159 (G), 436 (T), 437 (C), 451 (G), 453 (A), 485 (C); tef1 positions 46 (A), 62 (G), 80 (T), 100 (G), 146 (T), 274 (indel), 304 (A), 310 (G), 313 (C), 339 (T), 343 (A), 385 (G); twb2 positions 393 (A), 402 (indel), 426 (A), 565 (C), 675 (T), 713 (G), 770 (T). New polyketides from Diaporthe breyniae 97 Diaporthe amygdali CBS 126679" Diaporthe eres CBS 138594" Diaporthe siamensis MFLUCC 10-0573a Diaporthe yunnanensis CGMCC 3.18289" Diaporthe chinensis MFLUCC 19-01017 100/1 70/1 Diaporthe vitimegaspora STE-U2675 Diaporthe fici-septicae MFLU 18-2588" Diaporthe citriasiana CBS 134240 Diaporthe biguttulata ICMP 206577 -/0.97 99/1 1900/1 4100/1 Diaporthe discoidispora |CMP 20662° Diaporthe cinnamomi CFCC 52569° Diaporthe ambigua CBS 1140157 Diaporthe goulteri BRIP 55657a™ 6.67" Diaporthe sclerotioides CBS 296. Diaporthe longispora CBS 194.36" 4100/1 88/- 7T9/- Diaporthe mayteni CBS 133185" 74/1 100/1 Diaporthe tullilensis BRIP 62248a Diaporthe celtidis NCYU 19-0357" Diaporthe tectonae MFLUCC 12-0777" Diaporthe glabrae SCHM 36227 Diaporthe alangii CFCC 525567 japorthe hubeiensis JZB 3201237 Diaporthe cerradensis CMRP 43317 Diaporthe neoraonikayaporum MFLUCC 14-1136" Diaporthe raonikayaporum CBS 133182" 100/1 Diaporthe camporesii JZB 3201437 Diaporthe manihotia CBS 505.76 Diaporthe ganjae CBS 180.917 Diaporthe compacta LC 30837 Diaporthe sambucusii CFCC 51986' Diaporthe arezzoensis MFLU 19-2880" +1 100/41 -/0,98 -/0.98 Diaporthe myracrodruonis URM 79727 Diaporthe seratfiniae BRIP 55665a Diaporthe beilharziae BRIP 54792" Diaporthe infecunda CBS 133812" — Diaporthe pachirae COAD 7287 Diaporthe pyracanthae CBS 1423847 Diaporthe leucospermi CBS 1119807 100/1 Diaporthe rossmaniae CAA 762° 99/1, Diaporthe chimonanthi SCHM 3614" | 99/1] -/0.99 84/1 88/1 70/1 97/1 aun 99/1 99/1 -/0.98 0.04 Diaporthe micheliae SCHM 3603 Diaporthe acaciarum CBS 138862" Diaporthe middletonii BRIP 54884e" Diaporthe sackstonii BRIP 54669b" Diaporthe caryae CFCC 525637 Diaporthe machili SAUCC 194.1117 Diaporthe sinensis CGMCC 3.195217 Diaporthe lusitanicae CBS 1232127 Diaporthe cuppatea CBS 1174997 72/-,Diaporthe novem CBS 127271" 83/1. 'Diaporthe pseudolongicolla CBS 117165" 81/4 Diaporthe schoeni MFLU 15-12797 Diaporthe acericola MFLUCC 17-0956" Diaporthe neoarctii CBS 109490 Diaporthe cichorii MFLUCC 17-1023" Diaporthe guttulata CGMCC 3.20100° Diaporthe angelicae CBS 1115927 — Diaporthe subordinaria CBS 101711 Diaporthe arctii CBS 136.25 Diaporthe gulyae BRIP 54025" Diaporthe cucurbitae DAOM 420787 Diaporthe stewartii CBS 193.36 Diaporthe helianthi CBS 592.817 Diaporthe hordei CBS 481.92 Diaporthe vexans CBS 127.14 Diaporthe ata CBS 143.27 Diaporthe terebinthifolii CBS 1331807 Diaporthe tecomae CBS 100547 — Diaporthe racemosae CBS 1437707 Diaporthe schini CBS 1331817 Diaporthe rosiphthora COAD 2913° Diaporthe guangdongensis ZHKUCC 20-0014" Diaporthe melonis CBS 507.78" Diaporthe batatas CBS 122.21 Diaporthe convolvuli CBS 124654 Diaporthe ovalispora ICMP 206597 99/1 Diaporthe sojae CBS 1392827 Diaporthe kochmanii BRIP 54033" Diaporthe phaseolorum CBS 113425 Diaporthe endophytica CBS 1338117 83/1 Diaporthe fructicola MAFF 246408" 4. Diaporthe masirevicii BRIP 57892a™ 10.99 Diaporthe kongii BRIP 540317 10.93] Diaporthe subellipicola KUMCC 17.0153" “ _jy|Diaporthe chromolaenae MFLUCC 17-14227 Diaporthe heterostemmatis SAUCC 194.85" Diaporthe infertilis CBS 230.527 Diaporthe tectonendophytica MFLUCC 13-0471" Diaporthe longicolla FAU 599° Diaporthe unshiven Vi .) 92/1 74/0.99 100/1 SA /1 100/1 QT 92/1 95/1 95/1 Diaporthe thunbergiicola NV U -O0 Diaporthe rosae MFLUCC 17-2658" Diaporthe durionigena VTCC 930005" o6/ Diaporthe passifloricola CBS 141329° Diaporthe vochysiae LGMF 1583" 4 Diaporthe ueckeri BRIP 54736j (type strain of D. miriciae) 400/1+ Diaporthe ueckeri FAU 656 Figure |. ML (InL = -28100.2019) phylogram obtained from the combined ITS, cal, his3, tefl and tub2 sequences of our strain and related Diaporthe spp. Diaporthe amygdali CBS 126679! and D. eres CBS 138594" were used as an outgroup. Bootstrap support values > 70/Bayesian posterior probability scores > 0.95 are indicated along branches. Branch lengths are proportional to distance. New taxon is indicated in bold. Type material of the different species is indicated with *. 98 Blondelle Matio Kemkuignou et al. / MycoKeys 90: 85-118 (2022) Culture characters. Colonies on PDA reaching 55—70 mm in 2 weeks, greyed yel- low (161A) with a white ring and transparent margins, lobate, cottony, raised, margins filamentous to fimbriate; reverse greyed yellow (161A—D) with transparent margins. Colonies on MEA covering the surface of the Petri dish in 2 weeks, white with greyed yellow center (161A), velvety to cottony, flat to raised in some zones, margins fila- mentous to fimbriate; reverse greyed yellow (162A-—B). Colonies on OA covering the surface of the Petri dish in 2 weeks, white with greyed yellow ring (161D), velvety, flat, margins filamentous to fimbriate; reverse grey brown (199D). Specimen examined. Cameroon, Kala mountain, on leaves of Breynia oblongi- folia, 02 Jan. 2019, S.C_.N. Wouamba (holotype: CBS H-24920, culture ex-type CBS 148910 = STMA 18284). Notes. Diaporthe breyniae is introduced based only on molecular data since sporu- lation could not be induced in any media used. This species is located in a well-sup- ported clade (97% bs / 1 pp) together with D. durionigena, D. passifloricola, D. rosae, D. thunbergiicola, D. ueckeri and D. vochysiae. The latter species has only been reported from Brazil occurring on different hosts, i.e. Stryphnodendron adstringens (Fabaceae, Fa- bales) and Vochysia divergens (Vochysiaceae, Myrtales) (Noriler et al. 2019). Diaporthe durionigena has been only isolated from Durio zibethinus (Malvaceae, Malvales) in Vietnam (Crous et al. 2020, 2021). Diaporthe passifloricola has been found on Pas- siflora foetida (Passifloraceae, Malpighiales) and Citrus spp. (Rutaceae, Sapindales) in China and Malaysia (Crous et al. 2016; Chaisiri et al. 2021; Dong et al. 2021), while D. rosae has been isolated from Rosa sp. (Rosaceae, Rosales), Magnolia cham- paca (Magnoliaceae, Magnoliales) and Senna siamea (Fabaceae, Fabales) in Thailand (Perera et al. 2018; Wanasinghe et al. 2018). Diaporthe ueckeri (syn. D. miriciae, Gao et al. 2016) has been reported in Australia, Colombia and the USA, on Cucumis melo (Cucurbitaceae, Cucurbitales), Glycine max (Fabaceae, Fabales) and Helianthus annuus (Asteraceae, Asterales) (Thompson et al. 2015; Udayanga et al. 2015; Lopez-Cardona et al. 2021). Diaporthe thunbergiicola has been only isolated from Thunbergia laurifolia (Acanthaceae, Lamiales) in Thailand (Liu et al. 2015). The new species D. breyniae is the only of these species reported on Breynia (Phyllanthaceae, Malpighiales) in Africa. In fact, to the best of our knowledge, this is the first species of Diaporthe reported in Cameroon and occurring in this host. Structure elucidation of compounds I-13 Cultivation trials carried out on Diaporthe breyniae in different culture media includ- ing YM 6.3, Q6 %, ZM ¥, rice solid and YM agar highlighted its potential for produc- ing secondary metabolites. During antimicrobial screening of the extracts, the fungus revealed significant antifungal and antibacterial activity against Mucor hiemalis and Bacillus subtilis respectively, especially when cultured in ZM % medium, encouraging more detailed examination. Investigation into the chemistry of Diaporthe breyniae led to the isolation of two new secondary metabolites (7, 8) together with eleven known compounds (1-4, 5, 6, 9-13) from the EtOAc extracts of a 2 L scale-up ZM %% liquid New polyketides from Diaporthe breyniae 99 medium of the fungus (Fig. 2). The structure elucidation of 1-13 was determined by detailed spectroscopic analysis of their 1D and 2D NMR data in combination with their HR-ESIMS data. HR-ESI(+)MS and NMR spectroscopic analysis identified compounds 1-3 as cytochalasin H (1) (Suppl. material 1: Figs S3-S10) (Beno et al. 1977; Shang et al. 2017), deacetylcytochalasin H or cytochalasin J (2) (Suppl. material 1: Figs S11-S17) (Cole et al. 1981; Shang et al. 2017) and cytochalasin RKS-1778 (3) (Suppl. mate- rial 1: Figs S18-S24) (Kakeya et al. 1997) respectively. The absolute configuration of cytochalasins H (1) and J (2) was confirmed by comparing their optical rotation values ([a]?°,, +55.7 (c 0.158, MeOH) for 1 and [«]”*, +35.3 (c 0.394, MeOH) for 2) and ECD spectrum (Fig. 3) with those reported in the literature (Shang et al. 2017; Ma et al. 2021). The literature reports only the relative configuration of compound 3 (rel- (3S, 4R, 5S, 8S, 9S, 13£, 16S, 18R, 19F, 21R)) (Kakeya et al. 1997), therefore, its absolute configuration was investigated by comparison of its ECD spectrum with that of cytochalasins H (1) and J (2) (Fig. 3). The ECD spectrum of 3 showed negative ¢ 200 nm) cotton effect, the shape of which matched with that of compounds 1 and 2. Thus, the hitherto unestablished absolute configuration of cytochalasin RKS-1778 (3) was confirmed to be 3S, 4R, 5S, 8R, 9R, 13, 16S, 18R, 19F, 21R. HR-ESI (+) MS analysis of 4 isolated as a yellowish oil afforded pseudo-molecular ion peaks [M+H]* at m/z 436.2852 and [M+Na]* at m/z 458.2665 attributed to the molecular formula C,,H,,NO, (11 degrees of unsaturation). Comparison of the 1D and 2D NMR spectroscopic data for 4 (DMSO-d _) with those for 3 (Table 2) revealed that both compounds are closely related, with compound 4 being the deacetylated derivative of 3. This was confirmed on the 'H NMR spectrum of compound 4 by the absence of the methyl group H,-25 and on its '*C NMR spectrum by the absence of both C-24 carbonyl group and C-25 methyl group as visible on the NMR data recorded for compound 3 (Table 2). The relative configuration of compound 4 was determined by analysis of the coupling constants and NOESY correlations. The E- geometry of the A'*"* and A’??? double bonds in the macrocyclic ring was determined based on the large coupling constants / = 15.3 and 16.7 Hz observed between H-13 and H-14 and between H-19 and H-20 respectively. The small coupling constant / = 4.4 Hz observed between H-4 and H-5 confirmed their cis relationship (Kakeya et al. 1997). The NOESY spectrum arbitrarily suggested a-orientation of H-3, H-11, H-21 and H-23 based on the observed correlations between H-3/H-11, H-20/H-21 and H- 20/H-23,while the f-orientation of H-4, H-5, H-8, H-16, 18-OH and 21-OH were apparent from a network NOESY correlations between H-4/H-5, H-5/H-8, H-8/21- OH, 21-OH/H-19, H-19/H-16 and H-16/18-OH (Fig. 4). These correlations al- lowed the assignment of the relative configuration of compound 4 as either re/- (3S, 4R, 5S, 8S, 9S, 13E, 16S, 18R, 19F, 21R) or rel (BR, 4S, 5R, 8R, OR, 134, 16R, 185, 19F, 21S). In addition, the optical rotation value of 4 ([«]”°, -17.6 (c 0.278, MeOH)) approximating that reported in the literature for 3 ([u]*°,, -20 (c 0.05, MeOH, Kakeya et al. 1997) revealed that both compounds are levorotatory, and this suggested the stereochemistry of 4 to be identical to that of 3. The latter assumption was confirmed 100 Blondelle Matio Kemkuignou et al. / MycoKeys 90: 85-118 (2022) Figure 2. Chemical structures of compounds 1-13 isolated from Diaporthe breyniae. by comparing the ECD spectrum of 4 with those of compounds 1, 2 and 3. The same negative Cotton effect (200 nm) observed for all those compounds unambiguously certified the absolute configuration of compound 4 established as 3S, 4R, 5S, 8S, 9S, New polyketides from Diaporthe breyniae 101 293 343 393 wavelength [nm] 290 340) 390 wavelength [nm] Figure 3. ECD spectra of compounds 1-4 in MeOH. 13E, 16S, 18R, 19£, 21R. Thus, the structure of 4 was determined. This compound was regarded new while the current study has been under review, but concurrently it was published as phomopchalasin N by Chen et al. (2022). Interestingly, the authors also isolated it from a member of the genus Diaporthe, but inadvertently referred to their producer organism under the outdated name “Phomopsis”. We have decided to leave our complete data on the structure elucidation in the manuscript, so they can be compared with those of Chen et al. (2022) by other scientists, but the compounds are indeed identical. Compounds 5 and 6 were readily identified as the known fusaristatins A and B respectively, after careful analysis of their HR-ESI (+) MS and NMR spectroscopic data (Suppl. material 1: Figs S34—S47). Fusaristatins A (5) and B (6) were first reported in 2007 from an endophytic Fusarium sp. (Shiono et al. 2007) and so far, only fusa- ristatin A (5) has been isolated from D. phaeseolorum and D. longicolla (syn: Phomopsis longicolla) (Santos et al. 2011; Choi et al. 2013; Cui et al. 2017). Therefore, this is the first report for the isolation of fusaristatin B (6) from the genus Diaporthe. In addi- tion, two new derivatives of fusaristatin A (7, 8) were isolated from Diaporthe breyniae and their structures were established by intensive analysis of their 1D and 2D NMR spectroscopic data in combination with HR-ESIMS data and by comparison with the data reported in the literature for fusaristatins A (5) and B (6) (Shiono et al. 2007). The molecular formula of compound 7, isolated as a colorless oil, was determined to be C,,H.N,O, from the HR-ESIMS (positive mode) which showed pseudo-mo- lecular ion peaks [M+H]* at m/z 660.4219 and [M+Na]* at m/z 682.4024, indicating 10 degrees of unsaturation. Inspection of the molecular formula of 7 (C,.H.~N,O,) in comparison to that of 5 (C,,H.,N,O,) suggested that an amino group (-NH,) in compound 5 could probably have been replaced by a hydroxyl group (-OH) in com- pound 7. Intensive analysis of 1D and 2D NMR spectroscopic data (C,D,N) of com- pound 7 in comparison to that of 5 indicated that most signals in 7 were the same as those for 5 (Table 3), implying that 7 and 5 are closely related. The only difference was observed on the 'H NMR spectrum where the signal corresponding to the amino 102 Blondelle Matio Kemkuignou et al. / MycoKeys 90: 85-118 (2022) Table 2. °C (125 MHz) and 'H-NMR (500 MHz) spectroscopic data (DMSO-d, 6 in ppm) of com- pounds 3, 4. 3 4 No. dos type d,, J in Hz) Oc» type d,, Vin Hz) 1 174.3, C - 175.9, C - 2-NH - 7.89, s - 7.57, 8 3 53.9, CH 3.16, m 53.8, CH 3.14, q (4.9) 4 50.5, CH 2.02, t (4.1) 50.9, CH 2.47, t (4.4) 5 34.1, CH 2.18, m* 34.3, CH 2.3, m 6 137.3, C - 137.1, C - 7 126.8, CH 5.21* 127.4, CH 5.17, brs 8 42.3, CH 3.06 br d (9.9) 40.9, CH 3.04, br d (9.8) 9 55253. - S12 1C. : 10 44.0, CH, 2.59, dd (13.2, 7.4) 2.74, dd (13.1, 5.3) 43.6, CH, 2.65, dd (13.6, 5.2) 2.70, dd (13.6, 5.2) 11 12.8, CH, 0.64, d (7.2) 13.0, CH, 0.84, d (7.3) 12 19.2, CH, 1.62, s 19.3, CH, 1.63, s 13 129.2, CH 5.73, dd (15.7, 10.1) 129.7, CH 5.66, dd (15.3, 10.1) 14 133.5, CH 5.08, ddd (15.3, 10.9, 4.5) 132.8, CH 5.02, ddd (15.3, 11.0, 4.4) 15 42.1, CH, 1.57, m* 1.89, br dd (12.4, 4.3) 42.3, CH, 1.52, q (12.5) 1.84, br dd (12.5, 4.2) 16 27.6, CH 1.69, m 27.7, CH 1.69, m 17 53.1, CH 1.37, br dd (13.6, 3.2) 1.59, m* I3yl CH, 1.34, br dd (13.4, 3.3) 1.60, dd (13.6, 3.3) 18 72.1,C - 72:2,-€ - 19 137.3, CH 5.36, dd (16.6, 2.3) 136.2, CH 5.61, dd (16.7, 2.4) 20 125.1, CH 5.71, dd (16.9, 2.4) 130.7, CH 5.76, dd (16.7, 2.4) 21 75.7, CH 5.23* 73.7, CH 3.63, br s 22 25.8, CH, 0.94, d (7.3) 25.9; CE 0.93, d (7.1) 23 31.0, CH, 1.13, s 31.5, CH, 1.12, s 24 169.3, C - - - 25 20.2, CH, 2.18, s - - 1’ 136.8, C - 136.9, C - 2°16’ 129.6, CH (x2) 7.12, d (7.0) 129.8, CH (x2) 7.21* 3/15’ 127.9, CH (x2) 7.29, t (7.5) 127.7, CH (x2) 7.29, t (7.7) 4’ 126.0, CH 7.21, t (7.5) 126.0, CH 7.21" 18-OH - 4.36, s - 4.17, s 21-OH - - 4 4.88, br d (5.6) *overlapping signals, assignments were supported by HSQC and HMBC group 34-NH, (6, 8.34) in compound 5 was absent in compound 7 (Table 3). Moreo- ver, in the HMBC spectrum of 7, correlations from H-31 to C-30, H-31/H-32 to C-33 suggested the presence of a glutamic acid residue instead of a glutamine residue as observed in 5. Based on 'H-'H COSY, 'H-'8C HSQC and 'H-’°C HMBC experi- ments (Fig. 5), the signals of all protons and carbons in the molecule were unambigu- ously assigned and compound 7 was identified as a new derivative of fusaristatin A named fusaristatin G. Compound 8 was obtained as a white amorphous solid. The molecular formula was established as C,-H..N,O, on the basis of the pseudo-molecular ion peaks [M+H]* at m/z 661.4542 and [M+Na]* at m/z 683.4354 observed in the HR-ESI(+)MS, indicat- ing 9 double bond equivalents. The molecular formula of 8 (C,,H,,N,O,) compared to that of 5 (C,,.H,.N,O.) showed an increase of 2 Da suggesting that a reduction occurred in compound 5 to afford compound 8. This assumption was confirmed on New polyketides from Diaporthe breyniae 103 cosy — HMBC ~~ “A ROESY £ atae £ B-lace Figure 4. Selected 'H—'H COSY, NOESY and HMBC correlations of 4. the 'H NMR spectrum of 8 where the signals in the downfield region corresponding to H.-22’ (8,, 5.60) and H,-22’ (6,, 6.24) as observed in 5 were missing, but instead the signal in the upfield region corresponding to a methyl group H,-22’ at 5,, 1.65 was recorded (Table 3). Moreover, an additional signal observed on the 'H NMR of 8 attributable to the methine H-22 (8,, 4.89) further confirmed this assumption, in- dicating that the reduction of 5 occurred on the A”? double bond to afford 8. The reduction of the double bond A”**’ further justified the upfield shift of the nitrogen- bearing proton 21-NH, which resonated at d,, 8.15 in compound 8 instead of 6, 10.43 as in compound 5. In the HMBC spectrum, the correlations observed between H-22’ and C-22/C-23, H-22 and C-22’/C-23 confirmed the presence of an alanine residue instead of dehydroalanine residue as previously reported for 5 (Shiono et al. 2007). Finally, the unambiguous assignment of all proton and carbon signals in metabolite 8 was achieved based on 'H-'®C HSQC and 'H-'°C HMBC experiments, thus iden- tifying compound 8 as a new derivative of fusaristatin A, for which the trivial name fusaristatin H was assigned. Compounds 9-13 were respectively identified as phomoxanthones A (9) and B (10) (Isaka et al. 2001), dicerandrol B (11) (Wagenaar and Clardy 2001), phomo- chromenone C (12) (Ding et al. 2017; Wei et al. 2021), and diaporchromanone C (13) (Wei et al. 2021) by comparison of their HR-ESIMS and 1D and 2 D NMR spectroscopic data (Suppl. material 1: Figs S65—S99) with those reported in the litera- ture. Physico-chemical characteristic of compounds 4, 7 and 8 Phomopchalasin N (4): Yellowish oil. [«]’°, -17.6 (c 0.278, MeOH), UV (MeOH, c= 0.013 mg/mL) i, (loge) 202 (4.32) nm. CD (c = 2.83 x 10° M, MeOH) i, (Ae) 200 (-7.66) nm. HR-ESIMS m/z 458.2665 [M + Na]*, 77/z 893.5440 [2M + Na]*, m/z 871.5621 [2M + H]*, m/z 418.2746 [M + H - H,O]*, m/z 436.2852 [M + H]* (Calcd lis) on bce) eo INCE 436.2846), t, = 10.47 min. For NMR data ((H: 500 MHz, 3C: 125 28° 38 MHz, DMSO-d), see Table 2. 104 Blondelle Matio Kemkuignou et al. / MycoKeys 90: 85-118 (2022) Fusaristatin G (7): colorless oil. [a]? -8 (c 0.1, MeOH), UV (MeOH, c = 0.02 mg/mL) i, (log ¢) 201 (4.21), 283 (3.96) nm. HR-ESIMS m/z 682.4024 [M + Na]*, 7/z 1341.8157 [2M + NaJ*, m/z 1319.8354 [2M + H]*, m/z 642.4102 [M +H Table 3. '°C and 'H-NMR spectroscopic data (pyridine-d, d in ppm) of compounds 5, 7, 8. 5 7? 8 No. Oc, type d,, 7 in Hz) Oc, type d,, J in Hz) Oc, type d,, 7 in Hz) 1 14.7, CH, 0.88* 14.7,CH, ——0.87* 14.5, CH, 0.87, t (6.9)* 2 23.4, CH, 1.20~1.31, m* 23.4,CH, 1.20+1.31,m* 23.1,CH, —-1.20+1.31, m* 3 32.6, CH, 1,201.31, m* 32.6,CH, 1.20-1.31,m* 32.3, CH, 1.20-1.31, m* 4 27.7, CH, 1,201.31, m* 27.7,CH, 1.20-1.31,m* 27.4, CH, 1.20-1.31, m* 5 30.3, CH, 1.20~1.31, m* 30.3,CH, 1.20-1.31, m* 30.1, CH, 1.20-1.31, m* 6 37.5, CH, 1.09, m* 1.20-1.31, m* 37.5,CH, 1.09, m*1.20- 37.3,CH, 1.09, m* 1.20~1.31, m* 1.31, m* 7 33.2, CH 1.39, m* 33.2, CH 1.40, m* 32.9, CH 1.38, m* im 20.0, CH, 0.88* 20.0,CH, —0.88* 19.8,CH, 0.87, d (6.9)* 8 36.8, CH, 1.20~1.31* 1.40, m* 36.9,CH, 1.20-1.31,m* 36.6,CH, 1.20-1.31, m* 1.40, m* 1.40, m* 9 27.2, CH, 2.19, m* 27.2,CH, 2.18,m 27.0, CH, 2.21, m* 10 144.5, CH 6.03, br t (7.4) 144.5,CH 6.03, brt (7.2) 144.3, CH 6.01, t (7.4) 11 133.9, C - 140.0, C - 133.9, C - 11’ 12.6, CH, 1.83, s 12.7,CH, -1.83,s —«:12.5, CH, 1.85, s 12 148.4, CH 7.54, d (15.7) 148.3,CH 7.56,d (15.7) 148.2, CH 7.55, d (15.7) 13 123.7, CH 6.40, d (15.7) 123.8,CH 6.40,d (15.7) 123.6, CH 6.45, d (15.7) 14 203.8, C - 203.6, C - 204.1, C - 15 44.5, CH 2.84, m 44.6,CH 2.80~2.88,m* 44.6, CH 2.88, m 15’ 17.7, CH, 1.10, d (6.9) 17.6,CH, 1.10,d(6.9) 17.1, CH, 1.13, d (6.9) 16 28.5, CH, 1.57, m 1.93~2.00, m* 28.3,CH, 1.54,m1.93- 29.1,CH, 1.66, m 2.04, m* 2.00, m* 17 30.3, CH, 1.87, m 1.93~2.00, m* 30.2,CH, 1.84,m 1.93~ 31.3, CH, 1.97, m 2.04, m* 2.00, m* 18 77.3, CH 5.44, m 77.2, CH 5.48, m 77.6, CH 5.45, m 19 44.6, CH 3.03, quin (7.0) 44.5,CH 3.05, quin (7,0) 45.6, CH 2.95, m 19" 15.8, CH, 1.30, d (7.0)* 15.9,CH, 1.33, d(7.3)* 14.9, CH, 1.35, d (7.3) 20 173.9, C - 174.0, C - W/353.G - 21-NH - 10.43, s - 10.55, s - 8.15, brs 22 139.6, C - 139.8, C - 50.9, CH 4.89, m 22 114.6, 5.60, s 6.24, s 114.3, 5:59,86,22,5 173 "CH, 1.65, d (7.1) CH, CH, 23 165.2, C - 165.3, C 173.9, C - 24-NH - 7.81, brs - 7.88, br t (6.1) cs 7.96, brs 25 43.0, CH, 3.81, dt (13.5, 6.9) 3.92, dt (13.3, 4.9) 43.0, CH, 3.78, dt (13.5, 42.1, CH, 3.49, dt (13.6, 3.8) 6.7) 3.94, m 4.04, dt (13.5, 7.9) 26 42.7, CH 2.87, m 42.7, CH 2.92, m 42.8, CH 2.85, m 26’ 15.5, CH, 1.30, d (7.0)* 15.8,CH, 1.33,d(7.3)* 14.9, CH, 1.22, d (7.3) 27 175.0, C - 175.1,C - 175.4, C a 28-NH - 9.06, br d (7.5) - 9.11, br d (7.7) - 8.90, br d (7.7) 29 53.6, CH 5.13, dd (14.3, 7.6) 53.4, CH 5.18, m* 53.6, CH 5.06, dd (12.9, 6.2) 30 172:32€ - 172.4, C - 1725;.€ - 31 27.6,CH, 2.63, dt (13.7, 7.0) 2.69~2.77, m* —27.5,CH, 2.62, dt (13.8, 27.3, CH, 2.51, m 2.68-2.74, m* 6.9) 2.71, tt (13.8, 6.9) 32 32.8, CH, 2.69~2.77, m* 32.1,CH, 2.80~2.88,m* 32.7,CH, —_2.68~2.74, m* 33 175:7C - 176.1, C - 176.7, C - 34-NH, - 8.34, s - - - 8.32, br s “overlapping signals: assignments were supported by HSQC and HMBC, *'H 500 MH,,, °C 125 MHz; °'H 700 MH,, °C 175 MH, New polyketides from Diaporthe breyniae 105 Figure 5. Selected 'H—-'H COSY and HMBC correlations of 7. - H,O}*, m/z 660.4219 [M + H]* (Calcd for C, ..N;O,” 660.4218), ¢, = 14.80 min. For NMR data (!H: 700 MHz, °C: 175 MHz, C.H.N-d), see Table 3. Fusaristatin H (8): White amorphous solid. [a], +14 (c 0.03, MeOH), UV (MeOH, c = 0.02 mg/mL) i (log ¢) 201 (4.24), 283 (4.20) nm. HR-ESIMS m/z 683.4354 [M + Na]*, /z 1343.8820 [2M + Na]*, m/z 1321.9000 [2M + H]*, m/z 661.4542 [M + H]* (Caled for C,.H..N,O,* 661.4535), ¢, = 14.46 min. For NMR 36 61 4 data (‘H: 700 MHz, °C: 175 MHz, CjH.N-d), see Table 3. Biological activity The extracts obtained from the fungal culture in ZM ¥% exhibited activities against Bacillus subtilis with MIC values of 75 ug/mL for the supernatant’s extract and 2.3 ug/mL for the mycelial extract. These extracts were also active against Mucor plumbeus with respective MIC values of 150 and 37.5 yg/mL. Moreover, the purified com- pounds 1-7, 9, 10, 12, and 13 were subjected to antimicrobial assays against a panel of bacteria and fungi. The minimum inhibitory concentration (MIC) values showed that all compounds were active against at least one of the tested micro-organisms at concentration of 66.7 ug/mL (Table 4). Overall, the majority of the tested compounds exhibited weak to moderate activity. However, significant activity was noted for pho- moxanthones A (9) and B (10) against Bacillus subtilis. Both compounds inhibited the growth of the latter bacterium with a MIC value of 1.7 ug/mL, which turned out to be 5 times stronger than that of oxytetracyclin used as positive control. In addi- tion, their MIC value of 4.2 ug/mL against the Gram-positive bacterium S. aureus was 106 Blondelle Matio Kemkuignou et al. / MycoKeys 90: 85-118 (2022) quite considerable in comparison to that of the other tested compounds. This finding concurs well with previously published data which reported the antimicrobial activ- ity of xanthone derivatives isolated from Diaporthe spp. (Wagenaar and Clardy 2001; Elsasser et al. 2005; Lim et al. 2010). The antimicrobial activity of dicerandrol B (11), a closely related congener of phomoxanthones A (9) and B (10) was not investigated in the present work due to the low amount of available sample, however, its activity against B. subtilis and S. aureus has previously been reported (Wagenaar and Clardy 2001). The antimicrobial activity of compound 8 was not assessed due to the paucity of the sample. The cytotoxicity of all the isolated compounds except 11 was evaluated against a panel of mammalian cell lines. Eight compounds, 1-5 and 8-10 showed activity in this assay whereas the other isolated metabolites were inactive under test conditions (Table 5). The very significant activity exhibited by compounds 1-4 against all tested cancer cell lines were in agreement with previous studies which have reported cytochalasins as potent cytotoxins (Shang et al. 2017). However, when comparing the activity of the cytochalasin 4, which is the deacetylated derivative of 3, it was quite interesting to no- tice that 4 is significantly less toxic than 3 leading to the hypothesis that the presence of the acetyl group in 3 is an important structural element in the biological activity of the studied cytochalasins. The aforementioned assumption, was also observed when com- paring the cytotoxicity of compound 1 and 2. In effect, 2 is the deacetylated derivative of 1, and the latter was also found to be less toxic than 1. These results therefore give some hints in regards to the structure activity relationship (SAR) of the isolated cytocha- lasins, which will be tested further for their inhibitory effect on actin. In the same assay, compound 5 and 8 were found to be active against KB3.1 cell line with IC,, value of 10.63 and 30.3 uM respectively whereas compound 6 and 7 bearing the same core skel- eton did not show any activity. These results indicated that the cytotoxicity of this class of compounds might possibly be enhanced by the presence of an amide group (C-33) as Table 4. Minimum Inhibitory Concentrations (MIC) of compounds 1-7, 9-10, 12—13 against tested microorganisms. MIC (ug/mL) Test organisms 2 3 4 5 6 7 5 10 12 = 13 References Acinetobacter baumanii - - - - - - - - - - - 0.26° Bacillus subtilis - =) E67 66:72 6-7 LG.7 ~ G7 81.2 66.7 8.3° Candida albicans - - - - - - 66.7. - - - 16.6" Chromobacterium violaceum - - - - - - - - - - - 0.83° Escherichia coli - - - - - - - - - - 1.7° Mucor hiemalis 66.7 - 66.7 66.7 66.7 66.7 66.7 16.7 66.7 66.7 66.7 8.3" Mycobacterium smegmatis - - - - - - - 66.7 - - - i777 Pichia anomala - - - - - - - - - - - SB" Pseudomonas aeruginosa - - - - - - - - - - - 0.218 Rhodoturula glutinis 66.7 - - - - - - - - - - 4.2" Schizosaccharomyces pombe 16.7 66.7 66.7 66.7 - - - - 66.7 - - 8.3" Staphylococcus aureus - - 66.7 66.7 66.7 66.7 42 42 66.7 - 0.83° (-): No inhibition, ‘Ciprobay 2.54 mg/mL, *Gentamycin 1 mg/mL, ‘Kanamycin 1 mg/mL, "Nystatin 1 mg/mL, °Oxytetracyclin 1 mg/ mL. Starting concentration for antimicrobial assay were 66.7 pg/mL. New polyketides from Diaporthe breyniae 107 Table 5. Cytotoxic activity of compounds 1-10, 12-13. IC, (uM) Cell lines 1 2 3 4 5 6 7 8 9 10 12 13 Epothilone B KB3.1 0.064 0.33 1.7 5.8 10.6 - - 30.3 0.36 0.91 - - 6.5x10° L929 0.19 1.5 1.3 10.8 >30.4 - - - 1.06 5.6 - - 6.5x104 A431 0.085 0.33 14.3 11.0 12.0 n.t n.t n.t 0.04 0.17 n.t n.t 1.2x10% MCE-7 0.14 3.1 7.3 19.3. 7.44 n.t n.t n.t 0.02 0.36 n.t n.t 8.2x10° A549 0.16 0.73 3.1 10.3. 19.7 nt n.t nt 0.43 1.0 n.t n.t 6.1x10° SKOV-3 0.073 0.33 13.6 45.9 13.9 n.t n.t n.t 0.15 0.65 n.t n.t 2.9x10* PC-3 0.14 0.29 4.2 9.4 73 nt nt nt 1.1 9.7 n.t n.t 9.5x104 n.t: not tested, (-): no activity. Starting concentration for cytotoxicity assay was 37 pg/mL shown in 5 and 8 instead of a carboxylic acid as observed in 6 (C-34) and 7 (C-33). In addition, phomoxanthones A (9) and B (10), exhibited strong cytotoxic activities with half-maximal inhibitory concentrations (IC,,) in the range 0.02 — 9.7 .M. These results were in accordance with previous published cytotoxicity of dimeric tetrahydroxanthone derivatives against human epidermoid carcinoma (KB), human breast cancer (BC-1), mouse lymphoma (L5178Y), human ovarian carcinoma (A2780), and African monkey kidney fibroblast (Vero) cell lines among others (Isaka et al. 2001; Ronsberg et al. 2013). Conclusion The genus Diaporthe has been regarded for decades as a potential source for the pro- duction of diverse bioactive secondary metabolites. In the present study, we suggest the introduction of the new species D. breyniae isolated from the twigs of Breynia oblongi- folia in Cameroon. From the liquid culture of this fungus, two previously undescribed polyketides were isolated together with eleven known compounds. The isolated com- pounds showed weak to strong antimicrobial activities as well as moderate cytotoxic activities overall. These results demonstrated that it should certainly be worthwhile to explore untapped geographic area like the African tropics in general and Cameroon in particular for the discovery of new fungi and the isolation of novel secondary metabo- lites produced by these with significant biological activities. Acknowledgments We are grateful to W. Collisi for conducting the cytotoxicity assays, C. Kakoschke for recording NMR data and E. Surges for recording HPLC-MS data. The authors wish to thank V. Nana (National Herbarium of Cameroon) for the botanical identifications and S.C.N. Wouamba for the isolation of the strain CBS 148910. Financial support by a personal PhD stipend from the German Academic exchange service (DAAD) to B.M.K. is gratefully acknowledged (programme ID- 57440921). Y.M.E is grateful for the postdoctoral stipendium received from Alexander-von-Humboldt Foundation, 108 Blondelle Matio Kemkuignou et al. / MycoKeys 90: 85-118 (2022) Germany. We are also grateful to The World Academy of Sciences (TWAS) (grant 18-178 RG/CHE/AF/AC_G-FR 3240303654), and the Alexander von Humboldt Foundation (AvH) through the equipment subsidies (Ref 3.4 - 8151/20 002), the Research Group Linkage (grant IP-CMR-1121341) and the hub project CECANO- PROF (3.4-CMR-Hub). Furthermore, we are grateful to the Deutsche Forschungsge- meinschaft for a Research Unit grant “Cytolabs” (DFG-FOR-5170). References Alfaro ME, Zoller S, Lutzoni F (2003) Bayes or bootstrap. A simulation study comparing the performance of Bayesian Markov chain Monte Carlo sampling and bootstrapping in assess- ing phylogenetic confidence. Molecular Biology and Evolution 20(2): 255-266. https:// doi.org/10.1093/molbev/msg028 Becker K, Wongkanoun S, Wessel AC, Bills GE Stadler M, Luangsa-Ard JJ (2020a) Phyloge- netic and chemotaxonomic studies confirm the affinities of Stromatoneurospora phoenix to the coprophilous Xylariaceae. 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Chemistry (Weinheim an der Bergstrasse, Germany) 22(43): 15257-15260. https://doi.org/10.1002/chem.201603734 Zhang D, Gao £ Jakovli¢ I, Zou H, Zhang J, Li WX, Wang GT (2020) PhyloSuite: An inte- erated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Molecular Ecology Resources 20(1): 348-355. https://doi.org/10.1111/1755-0998.13096 Supplementary material | Figures S1-S100, Tables S$1-S5 Authors: Blondelle Matio Kemkuignou, Lena Schweizer, Christopher Lambert, Elodie Giséle M. Anoumedem, Simeon E Kouam, Marc Stadler, Yasmina Marin-Felix Data type: Docx file. Explanation note: The following are available online: 1D, 2D NMR, ESIMS and HR- ESIMS spectra of compounds 1-13; Fig $100, ML phylogram including our strain and type and reference strains of Diaporthe spp.; Table S1—S4, Information of the phylogenetic study; Alignment of the ITS, cal, his3, tef1, tub2 sequences used in the second phylogenetic study. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.90.8287 1.suppl1