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Research Article
New polyketides from the liquid culture of Diaporthe breyniae sp. nov. (Diaporthales, Diaporthaceae)
expand article infoBlondelle Matio Kemkuignou§, Lena Schweizer, Christopher Lambert§, Elodie Gisèle M. Anoumedem|, Simeon F. Kouam|, Marc Stadler§, Yasmina Marin-Felix§
‡ Department of Microbial Drugs, Helmholtz Centre for Infection Research (HZI) and German Centre for Infection Research (DZIF), Braunschweig, Germany
§ Technische Universität Braunschweig, Braunschweig, Germany
| University of Yaoundé I, Yaounde, Cameroon
Open Access

Abstract

During the course of a study on the biodiversity of endophytes from Cameroon, a fungal strain was isolated. A multigene phylogenetic inference using five DNA loci revealed that this strain represents an undescribed 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 structures 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 ElectroSpray 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 moderate cytotoxic activity against all tested cancer cell lines with IC50 values in the range of 5.8–45.9 µM. The latter was found to be less toxic than the other isolated cytochalasins (13) 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 IC50 value of 30.3 µM.

Graphical abstract

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 (Chepkirui 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 chemical space of the cytochalasins, in order to establish structure-activity relationships (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 (Zaghouani et al. 2016; Long et al. 2018). Moreover, most of the compounds that were reported 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 extracts derived from this strain and decided to study its secondary metabolites. The current 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 × 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 µg/mL streptomycin sulphate and incubated at 28 °C. After 10 days, fungal colonies 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, Missouri, 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 London (1966). The fungus was grown in 2% tap water agar supplemented with sterile pine needles (PNA; Smith et al. 1996) to induce sporulation.

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 gDNA Miniprep Kit EZ-10 Spin Column protocol (NBS Biologicals, Cambridgeshire, UK). The amplification of the ITS, cal, his3, tef1 and tub2 loci were performed according to White et al. (1990) (ITS), Carbone and Kohn (1999) (cal and tef1), Glass and Donaldson (1995) (his3 and tub2) and Crous et al. (2004) (his3). PCR products were purified and sequenced using Sanger Cycle Sequencing method at Microsynth Seqlab GmbH (Göttingen, Germany), 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 compare with, a first phylogenetic analysis was carried out based on the combination of the five loci sequences (ITS, cal, his3, tef1, 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 accurately) 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 maximum 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 calculation and automatic selection of the appropriate nucleotide exchange model using 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 126679T and D. eres CBS 138594T 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 IQTree 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 visually for congruence among the single locus trees. Concurrently, a second tree was 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 arriving 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 phylogenetic analysis are included in Supplementary material. Sequences retrieved from GenBank are indicated in Table 1 and Suppl. material 1: S4.

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 × 50 mm, 1.7 µm; Waters, Milford, USA) connected to an amaZon speed ESI-Iontrap-MS (Bruker, Billerica, MA, USA). HPLC parameters were set as follows: solvent A: H2O + 0.1% formic acid, solvent B: acetonitrile (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 Technologies, Santa Clara, USA; column 2.1 × 50 mm, 1.7 µm, C18 Acquity UPLC BEH (waters), solvent A: H2O +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, Germany) 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, 1H-NMR: 500 MHz and 13C-NMR: 125 MHz) and an Ascend 700 spectrometer with 5 mm TCI cryoprobe (Bruker, Billerica, MA/USA, 1H-NMR: 700 MHz and 13C-NMR: 175 MHz).

Table 1.

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 Isolates1 GenBank accession numbers2 References
ITS tub2 his3 tef1 cal
Diaporthe acaciarum CBS 138862T KP004460 KP004509 KP004504 - - Crous et al. (2014)
D. acericola MFLUCC 17-0956T KY964224 KY964074 - KY964180 KY964137 Dissanayake et al. (2017)
D. alangii CFCC 52556T MH121491 MH121573 MH121451 MH121533 MH121415 Yang et al. (2018)
D. ambigua CBS 114015T KC343010 KC343978 KC343494 KC343736 KC343252 Gomes et al. (2013)
D. amygdali CBS 126679T KC343022 KC343990 KC343506 KC343748 KC343264 Gomes et al. (2013)
D. angelicae CBS 111592T KC343026 KC343994 KC343511 KC343752 KC343268 Gomes et al. (2013)
D. arctii CBS 136.25 KC343031 KC343999 KC343515 KC343757 KC343273 Gomes et al. (2013)
D. arezzoensis MFLU 19-2880T MT185503 MT454055 - - - Li et al. (2020)
D. batatas CBS 122.21 KC343040 KC344008 KC343524 KC343766 KC343282 Gomes et al. (2013)
D. beilharziae BRIP 54792T JX862529 KF170921 - JX862535 - Thompson et al. (2015)
D. biguttulata ICMP 20657T KJ490582 KJ490403 KJ490524 KJ490461 - Huang et al. (2015)
D. breyniae CBS 148910T ON400846 ON409186 ON409187 ON409188 ON409189 Present study
D. camporesii JZB 320143T MN533805 MN561316 - - - Hyde et al. (2020)
D. caryae CFCC 52563T MH121498 MH121580 MH121458 MH121540 MH121422 Yang et al. (2018)
D. celtidis NCYU 19-0357T MW114346 MW148266 - MW192209 - Tennakoon et al. (2021)
D. cerradensis CMRP 4331T MN173198 MW751671 MW751663 MT311685 MW751655 Iantas et al. (2021)
D. chimonanthi SCHM 3614T AY622993 Chang et al. (2005)
D. chinensis MFLUCC 19-0101T MW187324 MW245013 - MW205017 MW294199 de Silva et al. (2021)
D. chromolaenae MFLUCC 17-1422T MH094275 - - - - Mapook et al. (2020)
D. cichorii MFLUCC 17-1023T KY964220 KY964104 - KY964176 KY964133 Dissanayake et al. (2017)
D. cinnamomi CFCC 52569T MH121504 MH121586 MH121464 MH121546 - Yang et al. (2018)
D. citriasiana CBS 134240T JQ954645 KC357459 MF418282 JQ954663 KC357491 Huang et al. (2013)
D. compacta LC3083T KP267854 KP293434 KP293508 KP267928 - Gao et al. (2016)
D. convolvuli CBS 124654 KC343054 KC344022 KC343538 KC343780 KC343296 Gomes et al. (2013)
D. cucurbitae DAOM 42078T KM453210 KP118848 KM453212 KM453211 - Udayanga et al. (2015)
D. cuppatea CBS 117499T AY339322 JX275420 KC343541 AY339354 JX197414 Van Rensburg et al. (2006)
D. discoidispora ICMP 20662T KJ490624 KJ490445 KJ490566 KJ490503 - Huang et al. (2015)
D. durionigena VTCC 930005T MN453530 MT276159 - MT276157 - Crous et al. (2020)
D. endophytica CBS 133811T KC343065 KC344033 KC343549 KC343791 KC343307 Gomes et al. (2013)
D. eres CBS 138594T KJ210529 KJ420799 KJ420850 KJ210550 KJ434999 Udayanga et al. (2014)
D. fici-septicae MFLU 18-2588T MW114348 MW148268 - MW192211 - Tennakoon et al. (2021)
D. fructicola MAFF 246408T LC342734 LC342736 LC342737 LC342735 LC342738 Crous et al. (2019)
D. ganjae CBS 180.91T KC343112 KC344080 KC343596 KC343838 KC343354 Gomes et al. (2013)
D. glabrae SCHM 3622T AY601918 - - - - Chang et al. (2005)
D. goulteri BRIP 55657aT KJ197290 KJ197270 - KJ197252 - Thompson et al. (2015)
D. guangdongensis ZHKUCC20-0014T MT355684 MT409292 - MT409338 MT409314 Dong et al. (2021)
D. gulyae BRIP 54025T JF431299 KJ197271 - JN645803 - Thompson et al. (2015)
D. guttulata CGMCC 3.20100T MT385950 MT424705 MW022491 MT424685 MW022470 Dissanayake et al. (2020)
D. helianthi CBS 592.81T KC343115 KC344083 KC343599 KC343841 JX197454 Gomes et al. (2013)
D. heterostemmatis SAUCC 194.85T MT822613 MT855810 MT855581 MT855925 MT855692 Sun et al. (2021)
D. hordei CBS 481.92 KC343120 KC344088 KC343604 KC343846 KC343362 Gomes et al. (2013)
D. hubeiensis JZB 320123T MK335809 MK500148 - MK523570 MK500235 Manawasinghe et al. 2019
D. infecunda CBS 133812T KC343126 KC344094 KC343610 KC343852 KC343368 Gomes et al. (2013)
D. infertilis CBS 230.52T KC343052 KC344020 KC343536 KC343778 KC343294 Guarnaccia and Crous (2017)
D. kochmanii BRIP 54033T JF431295 - - JN645809 - Thompson et al. (2011)
D. kongii BRIP 54031T JF431301 KJ197272 - JN645797 - Thompson et al. (2011)
D. leucospermi CBS 111980T JN712460 KY435673 KY435653 KY435632 KY435663 Crous et al. (2011c)
D. longicolla FAU 599T KJ590728 KJ610883 KJ659188 KJ590767 KJ612124 Udayanga et al. (2015)
D. longispora CBS 194.36T KC343135 KC344103 KC343619 KC343861 KC343377 Gomes et al. (2013)
D. lusitanicae CBS 123212T KC343136 KC344104 KC343620 KC343862 KC343378 Gomes et al. (2013)
D. machili SAUCC 194.111T MT822639 MT855836 MT855606 MT855951 MT855718 Huang et al. (2021)
D. manihotia CBS 505.76 KC343138 KC344106 KC343622 KC343864 KC343380 Gomes et al. (2013)
D. masirevicii BRIP 57892aT KJ197277 KJ197257 - KJ197239 - Thompson et al. (2015)
D. mayteni CBS 133185T KC343139 KC344107 KC343623 KC343865 KC343381 Gomes et al. (2013)
D. megalospora CBS 143.27 KC343140 KC344108 KC343624 KC343866 KC343382 Gomes et al. (2013)
D. melonis CBS 507.78T KC343142 KC344110 KC343626 KC343868 KC343384 Gomes et al. (2013)
D. micheliae SCHM 3603 AY620820 - - - - Chang et al. (2005)
D. middletonii BRIP 54884eT KJ197286 KJ197266 - KJ197248 - Thompson et al. (2015)
D. myracrodruonis URM 7972T MK205289 MK205291 - MK213408 MK205290 da Silva et al. (2019)
D. neoarctii CBS 109490 KC343145 KC344113 KC343629 KC343871 KC343387 Gomes et al. (2013)
D. neoraonikayaporum MFLUCC 14-1136T KU712449 KU743988 - KU749369 KU749356 Doilom et al. (2017)
D. novem CBS 127271T KC343157 KC344125 KC343641 KC343883 KC343399 Gomes et al. (2013)
D. ovalispora ICMP 20659T KJ490628 KJ490449 KJ490570 KJ490507 - Huang et al. (2015)
D. pachirae COAD 2074T MG559537 MG559541 - MG559539 MG559535 Milagres et al. (2018)
D. passifloricola CBS 141329T KX228292 KX228387 KX228367 - - Crous et al. (2016)
D. phaseolorum CBS 113425 KC343174 KC344142 KC343658 KC343900 KC343416 Gomes et al. (2013)
D. pseudolongicolla CBS 117165T DQ286285 - - DQ286259 - Petrović et al. (2018)
D. pyracanthae CBS142384T KY435635 KY435666 KY435645 KY435625 KY435656 Santos et al. (2017)
D. racemosae CBS 143770T MG600223 MG600227 MG600221 MG600225 MG600219 Marin-Felix et al. (2019)
D. raonikayaporum CBS 133182T KC343188 KC344156 KC343672 KC343914 KC343430 Gomes et al. (2013)
D. rosae MFLUCC 17-2658T MG828894 MG843878 - - MG829273 Wanasinghe et al. (2018)
D. rosiphthora COAD 2913T MT311196 - - MT313692 MT313690 Pereira et al. (2021)
D. rossmaniae CAA 762T MK792290 MK837914 MK871432 MK828063 MK883822 Hilário et al. (2020)
D. sackstonii BRIP 54669bT KJ197287 KJ197267 - KJ197249 - Thompson et al. (2015)
D. sambucusii CFCC 51986T KY852495 KY852511 KY852503 KY852507 KY852499 Yang et al. (2018)
D. schini CBS 133181T KC343191 KC344159 KC343675 KC343917 KC343433 Gomes et al. (2013)
D. schoeni MFLU 15-1279T KY964226 KY964109 - KY964182 KY964139 Dissanayake et al. (2017a)
D. sclerotioides CBS 296.67T KC343193 KC344161 KC343677 KC343919 KC343435 Gomes et al. (2013)
D. serafiniae BRIP 55665aT KJ197274 KJ197254 - KJ197236 - Thompson et al. (2015)
D. siamensis MFLUCC 10-0573a JQ619879 JX275429 - JX275393 - Udayanga et al. (2012)
D. sinensis CGMCC 3.19521T MK637451 MK660447 - MK660449 - Feng et al. (2019)
D. sojae CBS 139282T KJ590719 KJ610875 KJ659208 KJ590762 KJ612116 Udayanga et al. (2015)
D. stewartii CBS 193.36 FJ889448 - - GQ250324 - Santos et al. (2010)
D. subellipicola KUMCC 17-0153T MG746632 MG746634 - MG746633 - Hyde et al. (2018)
D. subordinaria CBS 101711 KC343213 KC344181 KC343697 KC343939 KC343455 Gomes et al. (2013)
D. tecomae CBS 100547 KC343215 KC344183 KC343699 KC343941 KC343457 Gomes et al.(2013)
D. tectonae MFLUCC 12-0777T KU712430 KU743977 - KU749359 KU749345 Doilom et al. (2017)
D. tectonendophytica MFLUCC 13-0471T KU712439 KU743986 - KU749367 KU749354 Doilom et al. (2017)
D. terebinthifolii CBS 133180T KC343216 KC344184 KC343700 KC343942 KC343458 Gomes et al. (2013)
D. thunbergiicola MFLUCC 12-0033T KP715097 - - KP715098 - Liu et al. (2015)
D. tulliensis BRIP 62248a KR936130 KR936132 - KR936133 - Crous et al. (2015)
D. ueckeri FAU 656 KJ590726 KJ610881 KJ659215 KJ590747 KJ612122 Huang et al. (2015)
BRIP 54736j (type of D. miriciae) KJ197283 KJ197263 - KJ197245 - Thompson et al. (2015)
D. unshiuensis CGMCC 3.17569T KJ490587 KJ490408 KJ490529 KJ490466 - Huang et al. (2015)
D. vexans CBS 127.14 KC343229 KC344197 KC343713 KC343955 KC343471 Gomes et al.(2013)
D. vitimegaspora STE-U 2675 AF230749 - - - - Mostert et al. (2001)
D. vochysiae LGMF 1583T MG976391 MK007527 MK033323 MK007526 MK007528 Noriler et al. (2019)
D. yunnanensis CGMCC 3.18289T KX986796 KX999228 KX999267 KX999188 KX999290 Gao et al. (2017)

Small-scale fermentation and extraction

The fungus was cultivated in three different liquid media (YM 6.3 medium: 10g/mL malt extract, 4g/mL, yeast extract, 4g/mL, D-glucose and pH = 6.3, Q6 ½ medium: 10 g/mL glycerin, 2.5 g/mL D-glucose, 5 g/mL cotton seed flour and pH = 7.2; ZM ½ medium: 5 g/mL molasses, 5 g/mL oatmeal, 1.5 g/mL D-glucose, 4 g/mL saccharose, 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, Düren, 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 sulphate. 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, KH2PO4 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 а YM agar plate were inoculated into a 250 ml Erlenmeyer flask containing 100 mL of YM 6.3 medium. The seed culture 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 ½ medium (Suppl. material 1: Figs S1, S2). Moreover, the extracts obtained from the fungal culture in ZM ½ were active against Bacillus subtilis 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 × 500 mL Erlenmeyer flasks containing 200 mL of ZM ½ 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 × 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 dissolved 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 (Büchi, Pure C-850, 2020, Switzerland). VP Nucleodur 100-5 C18ec column (150 × 40 mm, 7 µm: Machery-Nagel, Düren, Germany) was used as stationary phase. Deionized water (Milli-Q, Millipore, Schwalbach, Germany) supplemented 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 revealed that seven fractions constituted pure compounds. The compounds obtained from mycelial and supernatant extracts were combined according to their respective HPLC-ESIMS retention time and molecular weight. Compound 1 (55.2 mg, tR = 7.80 min) was obtained from both the mycelium and supernatant extracts as well as compounds 2 (10.9 mg, tR = 6.27 min), 3 (2.6 mg, tR = 11.42 min) and 4 (5.6 mg, tR = 9.49 min). Compounds 5 (3.6 mg, tR = 13.46 min), 11 (0.7 mg, tR = 12.11 min) and 12 (2.0 mg, tR = 3.83 min) were only isolated from the mycelial extract. Fractions F4 from both the mycelium and supernatant extracts were combined and purified using an Agilent Technologies 1200 Infinity Series semi-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 × 10 mm, 5 µm: Machery-Nagel, Düren, Germany) was used as stationary phase and the flow rate was 3 mL/min. These fractions afforded compound 13 (2.34 mg, tR = 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 (Büchi, Pure C-850, 2020, Switzerland). VP Nucleodur 100-5 C18ec column (250 × 21 mm, 5 µm: Machery-Nagel, Düren, 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, tR = 13.02 min) and sub-fraction G1. Sub-fraction G1 was further purified using 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, tR = 13.91 min) and 8 (0.52 mg, tR = 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, tR = 14.02 min) and 10 (1.7 mg, tR = 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 filamentous fungi and yeast and MHB medium (Müller-Hinton Broth: SN X927.1, Carl Roth GmbH, Karlsruhe, Germany) for bacteria. Starting concentration for all extracts were 300 µg/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 microorganisms, Bacillus subtilis DSM 10, Staphylococcus 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 µg/mL.

Cytotoxicity assay

The in vitro cytotoxicity (IC50) of the isolated metabolites against several mammalian cell lines (human endocervical adenocarcinoma KB 3.1, mouse fibroblasts L929, squamous 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 assay 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 (his3), 153 bp (tef1) 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 species 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.

Figure 1. 

ML (lnL = -28100.2019) phylogram obtained from the combined ITS, cal, his3, tef1 and tub2 sequences of our strain and related Diaporthe spp. Diaporthe amygdali CBS 126679T and D. eres CBS 138594T 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 T.

Taxonomy

Diaporthe breyniae Y. Marín & 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 phylogenetic 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); tub2 positions 393 (A), 402 (indel), 426 (A), 565 (C), 675 (T), 713 (G), 770 (T).

Culture characters

Colonies on PDA reaching 55–70 mm in 2 weeks, greyed yellow (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 filamentous 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 oblongifolia, 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 sporulation could not be induced in any media used. This species is located in a well-supported 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, Fabales) 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 Passiflora 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 champaca (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; López-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 1–13

Cultivation trials carried out on Diaporthe breyniae in different culture media including YM 6.3, Q6 ½, ZM ½, rice solid and YM agar highlighted its potential for producing 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 ½ 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 (14, 5, 6, 913) from the EtOAc extracts of a 2 L scale-up ZM ½ liquid medium of the fungus (Fig. 2). The structure elucidation of 113 was determined by detailed spectroscopic analysis of their 1D and 2D NMR data in combination with their HR-ESIMS data.

Figure 2. 

Chemical structures of compounds 113 isolated from Diaporthe breyniae.

HR-ESI(+)MS and NMR spectroscopic analysis identified compounds 13 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. material 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 ([α]20D +55.7 (c 0.158, MeOH) for 1 and [α]20D +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, 13E, 16S, 18R, 19E, 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, 13E, 16S, 18R, 19E, 21R.

Figure 3. 

ECD spectra of compounds 14 in MeOH.

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 C28H37NO3 (11 degrees of unsaturation). Comparison of the 1D and 2D NMR spectroscopic data for 4 (DMSO-d6) 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 1H NMR spectrum of compound 4 by the absence of the methyl group H3-25 and on its 13C 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 ∆13,14 and ∆19,20 double bonds in the macrocyclic ring was determined based on the large coupling constants J = 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 J = 4.4 Hz observed between H-4 and H-5 confirmed their cis relationship (Kakeya et al. 1997). The NOESY spectrum arbitrarily suggested α-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 β-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 allowed the assignment of the relative configuration of compound 4 as either rel- (3S, 4R, 5S, 8S, 9S, 13E, 16S, 18R, 19E, 21R) or rel- (3R, 4S, 5R, 8R, 9R, 13E, 16R, 18S, 19E, 21S). In addition, the optical rotation value of 4 ([α]20D -17.6 (c 0.278, MeOH)) approximating that reported in the literature for 3 ([α]20D -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 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, 13E, 16S, 18R, 19E, 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.

Table 2.

13C (125 MHz) and 1H-NMR (500 MHz) spectroscopic data (DMSO-d6, δ in ppm) of compounds 3, 4.

3 4
No. δ C, type δ H (J in Hz) δ C, type δ H (J in Hz)
1 174.3, C - 175.9, C -
2-NH - 7.89, s - 7.57, s
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, br s
8 42.3, CH 3.06 br d (9.9) 40.9, CH 3.04, br d (9.8)
9 55.5, C - 57.2, C -
10 44.0, CH2 2.59, dd (13.2, 7.4) 2.74, dd (13.1, 5.3) 43.6, CH2 2.65, dd (13.6, 5.2) 2.70, dd (13.6, 5.2)
11 12.8, CH3 0.64, d (7.2) 13.0, CH3 0.84, d (7.3)
12 19.2, CH3 1.62, s 19.3, CH3 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, CH2 1.57, m* 1.89, br dd (12.4, 4.3) 42.3, CH2 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* 53.1, CH2 1.34, br dd (13.4, 3.3) 1.60, dd (13.6, 3.3)
18 72.1, C - 72.2, C -
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, CH3 0.94, d (7.3) 25.9, CH3 0.93, d (7.1)
23 31.0, CH3 1.13, s 31.5, CH3 1.12, s
24 169.3, C - - -
25 20.2, CH3 2.18, s - -
136.8, C - 136.9, C -
2´/6´ 129.6, CH (x2) 7.12, d (7.0) 129.8, CH (x2) 7.21*
3´/5´ 127.9, CH (x2) 7.29, t (7.5) 127.7, CH (x2) 7.29, t (7.7)
126.0, CH 7.21, t (7.5) 126.0, CH 7.21*
18-OH - 4.36, s - 4.17, s
21-OH - - - 4.88, br d (5.6)
Figure 4. 

Selected 1H–1H COSY, NOESY and HMBC correlations of 4.

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 fusaristatin 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 addition, 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 C36H57N3O8 from the HR-ESIMS (positive mode) which showed pseudo-molecular 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 (C36H57N3O8) in comparison to that of 5 (C36H58N4O7) suggested that an amino group (-NH2) in compound 5 could probably have been replaced by a hydroxyl group (-OH) in compound 7. Intensive analysis of 1D and 2D NMR spectroscopic data (C5D5N) of compound 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 1H NMR spectrum where the signal corresponding to the amino group 34-NH2 (δH 8.34) in compound 5 was absent in compound 7 (Table 3). Moreover, 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 1H-1H COSY, 1H-13C HSQC and 1H-13C HMBC experiments (Fig. 5), the signals of all protons and carbons in the molecule were unambiguously assigned and compound 7 was identified as a new derivative of fusaristatin A named fusaristatin G.

Table 3.

13C and 1H-NMR spectroscopic data (pyridine-d5, δ in ppm) of compounds 5, 7, 8.

5a 7b 8b
No. δ C, type δ H (J in Hz) δ C, type δ H (J in Hz) δ C, type δ H (J in Hz)
1 14.7, CH3 0.88* 14.7, CH3 0.87* 14.5, CH3 0.87, t (6.9)*
2 23.4, CH2 1.20 ̴ 1.31, m* 23.4, CH2 1.20 ̴ 1.31, m* 23.1, CH2 1.20 ̴ 1.31, m*
3 32.6, CH2 1.20 ̴ 1.31, m* 32.6, CH2 1.20 ̴ 1.31, m* 32.3, CH2 1.20 ̴ 1.31, m*
4 27.7, CH2 1.20 ̴ 1.31, m* 27.7, CH2 1.20 ̴ 1.31, m* 27.4, CH2 1.20 ̴ 1.31, m*
5 30.3, CH2 1.20 ̴ 1.31, m* 30.3, CH2 1.20 ̴ 1.31, m* 30.1, CH2 1.20 ̴ 1.31, m*
6 37.5, CH2 1.09, m* 1.20-1.31, m* 37.5, CH2 1.09, m* 1.20 ̴ 1.31, m* 37.3, CH2 1.09, m* 1.20 ̴ 1.31, m*
7 33.2, CH 1.39, m* 33.2, CH 1.40, m* 32.9, CH 1.38, m*
20.0, CH3 0.88* 20.0, CH3 0.88* 19.8, CH3 0.87, d (6.9)*
8 36.8, CH2 1.20 ̴ 1.31* 1.40, m* 36.9, CH2 1.20 ̴ 1.31, m* 1.40, m* 36.6, CH2 1.20 ̴ 1.31, m* 1.40, m*
9 27.2, CH2 2.19, m* 27.2, CH2 2.18, m 27.0, CH2 2.21, m*
10 144.5, CH 6.03, br t (7.4) 144.5, CH 6.03, br t (7.2) 144.3, CH 6.01, t (7.4)
11 133.9, C - 140.0, C - 133.9, C -
11´ 12.6, CH3 1.83, s 12.7, CH3 1.83, s 12.5, CH3 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, CH3 1.10, d (6.9) 17.6, CH3 1.10, d (6.9) 17.1, CH3 1.13, d (6.9)
16 28.5, CH2 1.57, m 1.93 ̴ 2.00, m* 28.3, CH2 1.54, m 1.93 ̴ 2.00, m* 29.1, CH2 1.66, m 2.04, m*
17 30.3, CH2 1.87, m 1.93 ̴ 2.00, m* 30.2, CH2 1.84, m 1.93 ̴ 2.00, m* 31.3, CH2 1.97, m 2.04, 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, CH3 1.30, d (7.0)* 15.9, CH3 1.33, d (7.3)* 14.9, CH3 1.35, d (7.3)
20 173.9, C - 174.0, C - 173.5, C -
21-NH - 10.43, s - 10.55, s - 8.15, br s
22 139.6, C - 139.8, C - 50.9, CH 4.89, m
22´ 114.6, CH2 5.60, s 6.24, s 114.3, CH2 5.59, s 6.22, s 17.3, CH3 1.65, d (7.1)
23 165.2, C - 165.3, C 173.9, C -
24-NH - 7.81, br s - 7.88, br t (6.1) - 7.96, br s
25 43.0, CH2 3.81, dt (13.5, 6.9) 3.92, dt (13.3, 4.9) 43.0, CH2 3.78, dt (13.5, 6.7) 3.94, m 42.1, CH2 3.49, dt (13.6, 3.8) 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, CH3 1.30, d (7.0)* 15.8, CH3 1.33, d (7.3)* 14.9, CH3 1.22, d (7.3)
27 175.0, C - 175.1, C - 175.4, C -
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)
30 172.3, C - 172.4, C - 172.5, C -
31 27.6, CH2 2.63, dt (13.7, 7.0) 2.69 ̴ 2.77, m* 27.5, CH2 2.62, dt (13.8, 6.9) 2.71, tt (13.8, 6.9) 27.3, CH2 2.51, m 2.68 ̴ 2.74, m*
32 32.8, CH2 2.69 ̴ 2.77, m* 32.1, CH2 2.80 ̴ 2.88, m* 32.7, CH2 2.68 ̴ 2.74, m*
33 175.7, C - 176.1, C - 176.7, C -
34-NH2 - 8.34, s - - - 8.32, br s
Figure 5. 

Selected 1H–1H COSY and HMBC correlations of 7.

Compound 8 was obtained as a white amorphous solid. The molecular formula was established as C36H60N4O7 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, indicating 9 double bond equivalents. The molecular formula of 8 (C36H60N4O7) compared to that of 5 (C36H58N4O7) showed an increase of 2 Da suggesting that a reduction occurred in compound 5 to afford compound 8. This assumption was confirmed on the 1H NMR spectrum of 8 where the signals in the downfield region corresponding to Ha-22´ (δH 5.60) and Hb-22´ (δH 6.24) as observed in 5 were missing, but instead the signal in the upfield region corresponding to a methyl group H3-22´ at δH 1.65 was recorded (Table 3). Moreover, an additional signal observed on the 1H NMR of 8 attributable to the methine H-22 (δH 4.89) further confirmed this assumption, indicating that the reduction of 5 occurred on the ∆22-22´ double bond to afford 8. The reduction of the double bond ∆22-22´ further justified the upfield shift of the nitrogen-bearing proton 21-NH, which resonated at δH 8.15 in compound 8 instead of δH 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 1H-13C HSQC and 1H-13C HMBC experiments, thus identifying compound 8 as a new derivative of fusaristatin A, for which the trivial name fusaristatin H was assigned.

Compounds 913 were respectively identified as phomoxanthones A (9) and B (10) (Isaka et al. 2001), dicerandrol B (11) (Wagenaar and Clardy 2001), phomochromenone 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 literature.

Physico-chemical characteristic of compounds 4, 7 and 8

Phomopchalasin N (4): Yellowish oil. [α]20D -17.6 (c 0.278, MeOH), UV (MeOH, c = 0.013 mg/mL) λmax (log ε) 202 (4.32) nm. CD (c = 2.83 × 10-3 M, MeOH) λmax (Δε) 200 (-7.66) nm. HR-ESIMS m/z 458.2665 [M + Na]+, m/z 893.5440 [2M + Na]+, m/z 871.5621 [2M + H]+, m/z 418.2746 [M + H - H2O]+, m/z 436.2852 [M + H]+ (Calcd for C28H38NO3+ 436.2846), tR = 10.47 min. For NMR data (1H: 500 MHz, 13C: 125 MHz, DMSO-d6), see Table 2.

Fusaristatin G (7): colorless oil. [α]20D -8 (c 0.1, MeOH), UV (MeOH, c = 0.02 mg/mL) λmax (log ε) 201 (4.21), 283 (3.96) nm. HR-ESIMS m/z 682.4024 [M + Na]+, m/z 1341.8157 [2M + Na]+, m/z 1319.8354 [2M + H]+, m/z 642.4102 [M + H - H2O]+, m/z 660.4219 [M + H]+ (Calcd for C36H58N3O8+ 660.4218), tR = 14.80 min. For NMR data (1H: 700 MHz, 13C: 175 MHz, C5H5N-d5), see Table 3.

Fusaristatin H (8): White amorphous solid. [α]20D +14 (c 0.03, MeOH), UV (MeOH, c = 0.02 mg/mL) λmax (log ε) 201 (4.24), 283 (4.20) nm. HR-ESIMS m/z 683.4354 [M + Na]+, m/z 1343.8820 [2M + Na]+, m/z 1321.9000 [2M + H]+, m/z 661.4542 [M + H]+ (Calcd for C36H61N4O7+ 661.4535), tR = 14.46 min. For NMR data (1H: 700 MHz, 13C: 175 MHz, C5H5N-d5), see Table 3.

Biological activity

The extracts obtained from the fungal culture in ZM ½ exhibited activities against Bacillus subtilis with MIC values of 75 µg/mL for the supernatant´s extract and 2.3 µg/mL for the mycelial extract. These extracts were also active against Mucor plumbeus with respective MIC values of 150 and 37.5 µg/mL. Moreover, the purified compounds 17, 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 μg/mL (Table 4). Overall, the majority of the tested compounds exhibited weak to moderate activity. However, significant activity was noted for phomoxanthones A (9) and B (10) against Bacillus subtilis. Both compounds inhibited the growth of the latter bacterium with a MIC value of 1.7 μg/mL, which turned out to be 5 times stronger than that of oxytetracyclin used as positive control. In addition, their MIC value of 4.2 μg/mL against the Gram-positive bacterium S. aureus was quite considerable in comparison to that of the other tested compounds. This finding concurs well with previously published data which reported the antimicrobial activity of xanthone derivatives isolated from Diaporthe spp. (Wagenaar and Clardy 2001; Elsässer 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.

Table 4.

Minimum Inhibitory Concentrations (MIC) of compounds 17, 910, 1213 against tested microorganisms.

MIC (μg/mL)
Test organisms 1 2 3 4 5 6 7 9 10 12 13 References
Acinetobacter baumannii - - - - - - - - - - - 0.26c
Bacillus subtilis - - 16.7 66.7 16.7 16.7 1.7 1.7 66.7 8.3°
Candida albicans - - - - - - 66.7 - - - 16.6n
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 8.3n
Mycobacterium smegmatis - - - - - - - 66.7 - - - 1.7k
Pichia anomala - - - - - - - - - - - 8.3n
Pseudomonas aeruginosa - - - - - - - - - - - 0.21g
Rhodotorula glutinis 66.7 - - - - - - - - - - 4.2n
Schizosaccharomyces pombe 16.7 66.7 66.7 66.7 - - - - 66.7 - - 8.3n
Staphylococcus aureus - - 66.7 66.7 66.7 66.7 4.2 4.2 66.7 - 0.83°

The cytotoxicity of all the isolated compounds except 11 was evaluated against a panel of mammalian cell lines. Eight compounds, 15 and 810 showed activity in this assay whereas the other isolated metabolites were inactive under test conditions (Table 5). The very significant activity exhibited by compounds 14 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 notice 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 comparing 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 cytochalasins, 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 IC50 value of 10.63 and 30.3 µM respectively whereas compound 6 and 7 bearing the same core skeleton 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 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 (IC50) in the range 0.02 – 9.7 µM. 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; Rönsberg et al. 2013).

Table 5.

Cytotoxic activity of compounds 110, 1213.

IC50 (µM)
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 - - 6.5×10-5
L929 0.19 1.5 1.3 10.8 >30.4 - - - 1.06 5.6 - - 6.5×10-4
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 1.2×10-4
MCF-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 8.2×10-5
A549 0.16 0.73 3.1 10.3 19.7 n.t n.t n.t 0.43 1.0 n.t n.t 6.1×10-5
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 2.9×10-4
PC-3 0.14 0.29 4.2 9.4 7.3 n.t n.t n.t 1.1 9.7 n.t n.t 9.5×10-4

Conclusion

The genus Diaporthe has been regarded for decades as a potential source for the production 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 oblongifolia in Cameroon. From the liquid culture of this fungus, two previously undescribed polyketides were isolated together with eleven known compounds. The isolated compounds 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 metabolites 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.F. is grateful for the postdoctoral stipendium received from Alexander-von-Humboldt Foundation, 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 CECANOPROF (3.4-CMR-Hub). Furthermore, we are grateful to the Deutsche Forschungsgemeinschaft for a Research Unit grant “Cytolabs” (DFG-FOR-5170).

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Supplementary material

Supplementary material 1 

Figures S1–S100, Tables S1–S5

Blondelle Matio Kemkuignou, Lena Schweizer, Christopher Lambert, Elodie Gisèle M. Anoumedem, Simeon F. 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 113; Fig. S100, 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.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) 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.
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